Hormonal therapy for ovarian dysfunctions in high-productive cows

Liliya Khamitova
Izhevsk State Agricultural Academy
426069IzhevskRussia

Roman Rudakov
Izhevsk State Agricultural Academy
426069IzhevskRussia

Mariya Knyazeva
Izhevsk State Agricultural Academy
426069IzhevskRussia

Anastasiya Metlyakova
Izhevsk State Agricultural Academy
426069IzhevskRussia

Hormonal therapy for ovarian dysfunctions in high-productive cows
10.1051/bioconf/20201700205* Corresponding author:
The analysis was made of culling data in the farm of the Udmurt Republic. As a result, it was found that the animals are eliminated due to gynecological and limb diseases. They make up 19-24 % and 18-31 % respectively. Analyzing the results of gynecological clinical examination of cows, we found a predominance of ovarian diseases 32-36 %, as well as endometritis 28-30 %. The ovarian dysfunction progresses mostly. This can be attributed to an increase in the productivity of cows in the Udmurt Republic over the past 5 years from 5601 to 6250 kg per 1 cow. In connection with an increase in the incidence of ovaries, the calf yield per 100 cows decreases from 88 to 85 %. Causes of ovarian disease can be diseases of the birth and postpartum period. For the treatment of ovarian hypofunction, 3 schemes were used for anestral and reactive reproductive cycles. Often, the cause of ovarian hypofunction is uterine pathology; drugs that have a therapeutic effect on the uterus are included in the scheme.

Introduction

High productivity is accompanied by a violation of the reproductive function of cattle. Now this is the main problem of increasing animal productivity and the profitability of animal husbandry. From the barren cows of the economy, no offspring has been received; premature culling of cows occurs due to massive diseases of the uterus and ovaries. A large number of young cows are removed from the herd before costeffective use [1,2].

The ovaries are a paired organ with generative and endocrine functions [3]. The ovary is a dynamic organ, and its structure in the reproductive period undergoes constant cyclic changes [4].

Ovarian hypofunction (Hypofunctio ovarii) is a disease characterized by a decrease in the hormonal activity of the ovaries, in which inferior reproductive cycles (areactive, anestral, alibid, anovulatory) or anaphrodisia are observed [5].

Research purpose: to develop a therapy system for ovarian hypofunction at the Rico-Agro farm in the Uvinsky district of the Udmurt Republic.

To achieve this goal, the following tasks have been developed:

• To study the prevalence of ovarian dysfunction in cows in a particular farm (clinical and instrumental research, ultrasound);

• To study the therapeutic efficacy of hormonal drugs for the treatment of ovarian hypofunction in cows;

Materials

The work was carried out on the basis of the farm of Rico-Agro, in the Udmurt Republic, Uvinsky district, the village of Porshur-Tuklya. The farm is a breeding factory and is engaged in the cultivation of cattle of black and motley breed. The object of research is cattle, cows of black-motley breed, unborn cows with a service period of more than 60 days and a productivity of more than 6000 kg for 305 days of lactation.

Cows were selected by the method of pair-analogues according to the results of gynecological medical examination. They were selected according to the following characteristics: absence of reproductive cycles, violation of cycles, ovarian pathology.

To study the condition of animals on the farm, they used: retrospective, anamnestic, clinical research, including rectal research, ultrasound of the genital organs.

Animals for the experiment were selected for 10 animals with 2 and 3 calving and productivity of over 6000 kg for 305 days of lactation, as well as 5 heifers with a productivity of over 20 liters per day. The live weight of cows is over 550 kg, and the first-calf is over 500 kg.

Results

The farm contains 1646 heads of cattle of black and motley breed. Of these, 637 cows are cows. The average milk yield of cows for 305 days of lactation is 6250 kg. The yield of live calves is 85 %. This indicator over 5 years amounted to more than 88 %.

The Rico-Agro enterprise raises cattle of the dairy direction of a black and motley breed. They use artificial insemination of cows and heifers in a rectocervical way and sperm of outstanding bulls for milk productivity, mass fraction of fat and protein, which have categories for milk yield and mass fraction of fat. Sperm is acquired at specialized breeding enterprises.

Animals are kept in clean, dry, warm cowsheds with good lighting and ventilation. Cows have predominantly average fatness.

A stall-walking system is applied for keeping animals. Cattle are kept in rooms where both harness and free placement are used. Milking of animals takes place automatically in the milk pipeline on the ground and in the milking parlor on the installation. Manure is cleaned mechanically with a scraper conveyor.

From the main herd, cows are transferred to the maternity ward 10 to 20 days before calving. In the room, a leash is used, except for calving boxes, where animals are kept freely. Straw, hay are used as litter in the boxes. Boxes are disinfected after each calving, as are the stalls where new-born animals are located. Disinfection is carried out chemically after mechanical cleaning, during a passive exercise of animals daily.

Calves are kept for up to two days in calving with their mother, then transferred to a dispensary, and cows to a group of animals after calving. Milking occurs using a machine in a milk line. Manure is removed using a scraper conveyor.

Preventive clinics in winter are located in a building divided into two partsa barn and a calf. In summer, calves are kept in individual houses with walking yards. Animals are kept in this room for 20-30 days, then transferred to another calf, where animals are kept for up to 6 months. Milk calves are fed from a nipple drinker, and calves over 10 days of agefrom a bucket. In the winter period, calves are kept in group cages of 5-6 animals each. Cleaning of litter and manure is carried out manually every day.

Animals up to 6 months old are kept in cages, in groups of 10-15 animals each. Feeding is carried out from group feeders twice a daya substitute for whole milk, in free access are concentrates, hay, chalk; from 3-4 months old, they begin to add silage to the diet. Near the calf, there is a walking yard where passive exercise is carried out. Manure is cleaned manually and mechanically using a scraper conveyor.

The exercise is present in the main livestock, calves up to 6 months old, but in the winter-stall period they do not receive a full-fledged daily exercise, because the area of walking areas does not allow to drive out all animals every day. Newly born animals receive a daily passive exercise.

Silage-silage type of feeding is applied, feed is distributed using a mixer. Also, additives are usedprotein-mineral vitamin premix, selenium and kayod preparations, since the Udmurt Republic is located in the biogeochemical zone with the insufficient content of selenium and iodine in soils and water. Feeding in the winter-stall period is carried out twice a day. The total nutritional value of the diet of lactating cows averages 21.7 feed units. In the summer-pasture period, the diet of lactating cows includes: green mass, concentrates, salt and additives. For each liter of milk, 0.2 kg of concentrates are added to cows. Individual drinking bowls are used for drinking. In the chemical study of feed, the following results were obtained: hay and silage of the 1st -2nd class, haylage -the 2nd class. Cows are often rejected on the farm due to limb diseases. Laminitis, arthritis and sprain of limbs were revealed. Cows with such diseases often have ovarian hypofunction and uterine atony, and as a result the absence of reproductive cycles. This results from the inactivity of the animal.

For various reasons, gynecological diseases arise that prevent estrus, ovulation and conception in the first phase of development. The drugs used in the treatment of ovarian hypofunction are necessary for the full restoration of their function.

Surfagon is a hormonal drug, which contains the active substance luliberin acetate. It acts on the cells of the anterior pituitary gland, quickly reaching the maximum concentration in the blood. It is necessary to accelerate the maturation of follicles in the ovaries.

Fertagon is a hormonal drug containing in its composition such active substances as dalargin and alarelin acetate. Dalargin weakens the effect of stress on the body, and meanwhile, allarelin acetate stimulates follicular maturation and ovulation.

Estrofan is a prostaglandin that has a luteolytic effect on the corpus luteum of the ovaries, thereby reducing the amount of progesterone, which inhibits the growth of follicles.

Drugs exert their effect directly on the growth and development of follicles, while the effect on the uterus is very low or absent altogether. Due to the fact that postpartum complications, endometritis, atony and uterine subinvolution are a common cause of ovarian hypofunction, it is necessary to prevent these causes of the disease. Analysis of table 1 confirms the presence of these diseases.

There are problems on the farm: retention of the placenta, endometritis, subinvolution, atony of the uterus. On average, the retention of the placenta in a cow lasts 5-7 days and then when the cervix is opened, it is removed and treatment of endometritis begins.

In order to prevent retention of the placenta 6 hours after calving, cows are injected, depending on body weight, 2-3 ml of estrofan. To improve the recovery period, cows are fed energy shakes. In addition, in the first days after calving, cows are fortified with tetravit in a dose of 10 ml.

Immediately after removal of the placenta, the cows are given an intrauterine foam-forming tablet called «Enroflon», they are repeated every other day, and then treatment of the catarrhal-purulent endometritis is started by introducing the drug «Floxavet» into the uterine cavity, this procedure is repeated 3 times every other day.

Thus, the cow that has undergone this course of treatment in full by the 20th day after calving, recovered in 80 % of cases. The remaining animals in the amount of 20 % more often go into the chronic form of the course of endometritis. Of the obvious forms of endometritis, purulent-catarrhal prevails. It manifests itself with abundant purulent efflux from the genitals.

For the treatment of latent forms of uterine diseases, drugs are needed that provide a therapeutic effect, as well as increase its tone and contribute to the full passage of all stages of the sexual cycle.

For this experiment, drugs acting on the myometrium were selected: «Metrostim» and «Metrilong».

Metrostim, which includes carbacholine, briefly enhances the contraction of the smooth muscles of the uterus. The metrilong containing propranolol in the composition promotes the manifestation of the activity of endogenous oxytocin, thereby exerting a contracting effect on the smooth muscles of the uterus. Chitosan and glycerin, which are part of it, have a prolonging effect of propranolol.

Obstetric and gynecological medical examination is carried out on 15, 30 and 45 days after calving, in order to timely detect diseases of the reproductive system and conduct fruitful insemination the first time. Insemination of cows is carried out no earlier than 60 days after calving. The heifers are inseminated at the age of 15 months, upon reaching a live weight of not less than 360 kg.

There is no exercise on the farm, so the restoration of cows after calving forces the use of uterine-reducing drugs for a long time. At the same time, there is no highquality feed base, with year-round feeding of silos and haylage.

In the stall, sexual hunting is determined by changing the behaviour of the animal and the presence of outflows from the genitals. Before insemination, the spotted cow is checked rectally with an ultrasound scanner or by palpation for the presence and size of the follicle. The insemination technician conducts artificial insemination of cows in the rectocervical method, in the presence of a mature follicle. Cows are inseminated with sperm after thawing, as well as with a sexed seed, to increase the breeding stock of cows.

Thus, when developing these treatment regimens, we took into account the need for a combination of drugs for an integrated approach to treating ovarian hypofunction and increasing the reproductive ability of cows on the farm.

The production indicators of animals in the experiment are presented in table 2.

In connection with increasing the productivity of animals, problems with the reproductive ability of cows begin. This is evidenced by the annual decrease in calf yield. It is also worth noting a sharp jump in the disposal of cows from 10-12 to 15 %.

Gynecological diseases are one of the common causes of cows dropping out of the herd for several years, they occupy almost a third of all causes.

A significant number of cows are prone to ovarian disease. Sick animals are of different ages.   Table 3 shows the annual growth of ovarian disease. They occupy a third of all other gynecological diseases. The most common diseases of the ovaries are hypofunction of the ovaries and follicular cysts ( Table 4).

The main etiological factors that provoked the development of ovarian hypofunction are the lack of exercise in the postpartum period, and its incomplete provision, especially in the winterstall period, as well as intensive exploitation of animals [6,7,8].

The exercise has a positive effect on the cardiovascular, respiratory, digestive, sexual and musculoskeletal systems of cows [9]. Under conditions of physical inactivity, most animals after childbirth remain with impaired sexual function and infertile [10]. According to the results of an obstetric-gynecological study, 24 animals with a presumptive diagnosis of ovarian hypofunction were selected. Two animals of the first, second and third lactation were selected for the study.

Ovarian therapy in the household was previously carried out with standard drugs. For the treatment of follicular cysts, surfacton was used in a dose of 5 ml for 3 days, and for the treatment of hypofunction of the ovaries, the follimage was administered once in a dose of 1000 IU, and on day 14, estrofan in a dose of 2 ml. But, due to the fact that the follimage does not give the expected results, it is necessary to develop a new treatment regimen for ovarian hypofunction.

The developed treatment regimens for ovarian hypofunction are presented in table 5. These regimens are used for different forms of ovarian hypofunction. Scheme No. 1 and No. 2 are with anestral or arrhythmic sexual cycle; scheme No. 3 is with a full and rhythmic sexual cycle. The old treatment regime was used as a control.   3. 80 % of the tested animals according to these schemes became fruitful after the first insemination.

Gynecological diseases are very common in the reproduction of a herd of black-motley cattle. The farm "Rico-Agro" is no exception and has very high rates of incidence of cows in gynecology, and accordingly, the rates of reproduction are reduced. So from 2014 to 2018, culling for gynecological diseases progressed from 20.4 to 23.9 %, and ovarian diseases increased from 33-34 to 36 % over the past year. Based on this, the calf yield per 100 cows decreased from 88 to 85 %.

The main reasons for the decrease in reproductive ability are inflammatory processes of the genitals, lack of exercise, high productivity, lack of trace elements, violation of animal health standards, unbalanced diets, which violates metabolism, lack of energy, and a long stall period.

Thus, using treatment regimens No. 1 and 3, which include drugs such as Metrostim, Surfagon and Estrofan, we found high rates of cow fertilization from the first insemination. This indicator was 80 %. The use of new treatment regimens will help the farm to bring reproduction to a new level, with an intensive increase in indicators for artificial insemination of cows, a reduction in the service period and the insemination index.

Conclusion

High rates of milk productivity are increasingly accompanied by a malfunction of the reproductive system of cattle. This problem reduces the economic performance of production. Diseases of the ovaries and uterus are massive. Young cows are heavily rejected for cost-effective use. Ovarian hypofunction (Hypofunctio ovarii) is a dysfunctional disease characterized by a decrease in the hormonal activity of the ovaries, in which inferior reproductive cycles are observed, including the reactive and anestral forms. Complex therapy in the initial stage of the disease against the background of balanced feeding and improvement of the living conditions has a beneficial therapeutic effect. As a result of the experiment, positive dynamics with identical results were obtained using scheme No. 1 in the reactive cycle and scheme No. 3 in the anestral cycle. In general, fertilization occurred during the first insemination in 5 out of 6 cows. The preparation Metrostim (the active substance is carbacholine) has a restoring effect on the myometrium, and Surfagon, a synthetic analogue of GnRH, has a gonadotropic effect.

Table 1 .
1Removing cows from the herdCauses
year
2014
2015
2016
2017
2018
%
%
%
%
%
Low
productivity

22.2
19.6
20.7
21.0
15.6

Gynecological
diseases

20.4
19.6
19.0
21.0
23.9

Diseases of the
extremities

25.9
25.4
18.5
30.9
28.1

Trauma
7.4
9.8
6.9
4.9
6.2
Udder diseases
20.4
19.6
19.0
14.8
17.7
other diseases
3.7
5.9
8.6
7.4
8.3

Table 2 .
2Degree of damage to animals by gynecological diseasesGroups
of
animals

service period (days),
productivity (kg)
3 lactation
2 lactation
1 lactation
1
110,
6815

180,
7358

149,
6873

235,
6102

138
123

2
112,
7436

212,
7108

161,
6007

214,
7234

135
131

3
128,
7218

167,
7954

168,
7215

208,
6418

141
136

the
control

114,
7087

187,
6984

156,
6352

228,
7123

144
130

Table 3 .
3Degree of damage to animals by gynecological diseasesIndicators
Years
2014 2015 2016 2017 2018
gynecologically sick,
total: heads (%)

277
(100)

298
(100)

302
(100)

372
(100)

390
(100)
of hich: endometritis
heads (%)

79
(28)

85
(29)

91
(30)

107
(29)

108
(28)
retention of
membranes fetal heads
(%)

71
(26)

74
(25)

79
(26)

98
(26)

91
(23)

ovarian disease,
heads (%)

94
(34)

99
(33)

95
(32)

124
(33)

142
(36)
other gynecological
diseases, heads (%)

33
(12)

40
(13)

37
(12)

43
(12)

49
(13)

Table 4 .
4Degree of damage to cows by ovarian diseaseOvarian Diseases /
unit of measurement

2014 2015 2016 2017 2018

follicular cyst heads
28 31 29 37
40
luteal cyst heads
13 16 17 20
21
Hypofunction, heads
26 25 26 38
46
Atrophy of the ovaries, heads
2
1
4
3
3
persistent corpus luteum,
heads

21 26 19 26
32

Table 5 .
5Treatment regimens for ovarian hypofunctionScheme
№№

7 day cycle
14 day cycle
18 day cycle
21 day cycle
lack of a hunting
stage

18 day cycle

1
metrostim, 2 ml, 3
times in one day

Surfagon
10 ml 3 days

estrofan
2 ml once

insemination
estrofan
2 ml once
2
metrilong,
10 ml once

Fertagon
10 ml 3 days

estrofan
2 ml once

insemination
estrofan
2 ml once
3
metrostim, 2 ml, 3
times in one day

Surfagon
10 ml 3 days

-
Surfagon, 10 ml
1 hour before
insemination

-

the control
follimag, 1000
IU, once

estrofan
2 ml once

-
-
-

Table 6 .
6Ultrasound Results Ultrasound, in table 4, shows the positive dynamics of therapy according to schemes No. 1 and No.Scheme
№№

38 days after
the first
insemination,
heads

38 days after
the second
insemination,
heads

Not pregnant
60 days after
insemination,
heads

no
sex
hunt,
heads
pregnant cows, heads
1
5
-
1
-
2
2
1
2
1
3
5
-
1
-

© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/).
BIO Web of Conferences 17, 00205 (2020) https://doi.org/10.1051/bioconf/20201700205 FIES 2019

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Composite Effects of Polymorphisms near Multiple Regulatory Elements Create a Major-Effect QTL
2011

R D Bickel
A Kopp
S V Nuzhdin
Composite Effects of Polymorphisms near Multiple Regulatory Elements Create a Major-Effect QTL

PLoS Genet
7110012752011Received August 20, 2010; Accepted December 13, 2010;Editor: Gregory S. Barsh, Stanford University, United States of America The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. *
Many agriculturally, evolutionarily, and medically important characters vary in a quantitative fashion. Unfortunately, the genes and sequence variants accounting for this variation remain largely unknown due to a variety of biological and technical challenges. Drosophila melanogaster contains high levels of sequence variation and low linkage disequilibrium, allowing us to dissect the effects of many causative variants within a single locus. Here, we take advantage of these features to identify and characterize the sequence polymorphisms that comprise major effect QTL alleles segregating at the bric-abrac locus. We show that natural bric-a-brac alleles with large effects on cuticular pigmentation reflect a cumulative impact of polymorphisms that affect three functional regions: a promoter, a tissue-specific enhancer, and a Polycomb response element. Analysis of allele-specific expression at the bric-a-brac locus confirms that these polymorphisms modulate transcription at the cis-regulatory level. Our results establish that a single QTL can act through a confluence of multiple molecular mechanisms and that sequence variation in regions flanking experimentally validated functional elements can have significant quantitative effects on transcriptional activity and phenotype. These findings have important design and conceptual implications for basic and medical genomics.

Introduction

Quantitative trait loci (QTLs) have traditionally been identified by linkage analysis in experimental crosses [1]. In a complementary approach, DNA polymorphisms causing trait variation can be inferred from statistical associations between sequence and phenotype in large panels of natural genotypes [2]. These methods have been advanced with creative applications [3,4], but in most cases QTL regions have only been narrowed to a single gene. Understanding the identity, distribution, and functional nature of nucleotide polymorphisms that make up QTL alleles remains a critical challenge in molecular and evolutionary genetics. In this report, we take a QTL study to its logical conclusion by describing the fine structure of functional variation that underlies large-effect QTL alleles segregating in a natural population.

To meet this aim, we sequenced the 148 kb bric a brac (bab) genomic region from 94 inbred lines of D. melanogaster sampled from a single location. The bab locus is composed of two anciently duplicated paralogs, bab1 and bab2, which are involved in patterning the adult abdomen, legs, and ovaries [5]. In particular, Bab expression in the pupal abdominal epidermis is responsible for the sex-specific pigmentation of adult cuticle, and evolutionary changes in bab regulation have played a central role in the macroevolutionary diversification of Drosophila color patterns [6][7][8]. Moreover, segregation of bab alleles is responsible for ,60% of the genetic variation in female abdominal pigmentation within a natural population of D. melanogaster [9] (Figure 1). Linkage disequilibrium (LD) at the bab locus extends for less than 1 kb, [10] providing an opportunity to dissect the causative nucleotide variation that contributes to the cumulative effect of QTL alleles. Previous analysis revealed evidence of directional selection in two regions within the bab locus, including the previously identified cis-regulatory element (CRE) that controls sex-specific abdominal pigmentation [8,10]. This confluence of population-and developmental-genetic evidence sets the stage for connecting specific DNA polymorphisms to variation in gene expression and, ultimately, in phenotypic traits. We have sequenced the bab locus from 96 D. melanogaster lines to identify the genetic variation responsible for pigmentation differences.

Results/Discussion

Causative polymorphisms cluster in three functional regions

We identified 6766 sequence variants, including 5566 single nucleotide polymorphisms (SNPs) and 1211 insertion/deletion (indel) polymorphisms, in the bab region. After removing polymorphisms with frequencies below 5%, which are difficult to associate with phenotypic variation, 3821 polymorphism (SNPs and indels) remained. We tested each polymorphism individually for an association with female pigmentation, and identified 266 polymorphisms that showed significant association at the false discovery rate (FDR) ,0.4 (p,.033) (Spearman rank correlation, qvalue correction) [11] (Figure 2A). All associated polymorphisms are located in non-coding DNA.

To determine whether the significant polymorphisms were randomly distributed across the locus or clustered into functional regions, we looked at the proportion of associated polymorphisms in 5 kb sliding windows ( Figure 2B) (see Materials and Methods for details). We found that most associated polymorphisms are located in three clusters: a 15 kb region in the first intron of bab1, an 8 kb region between the bab1 and bab2 coding regions, and a 9.5 kb region near the transcription start site of bab2.

These clusters of significantly associated points could be the result of a high number of causative polymorphisms or alternatively a single causative polymorphism with high levels of LD resulting in a cluster of associated polymorphisms. We examined the pairwise levels of LD for significant polymorphisms within each region (see Figure S1). Based on the LD pattern, we conservatively estimate that there are at least 28 associated polymorphisms, or groups of polymorphisms, assorting independently in region 1, 8 polymorphisms in region 2 and 14 polymorphisms in region 3. Thus, there are multiple polymorphisms within each region that are likely to be causative variation.

The first region resides within the large first intron of bab1 and contains the cis-regulatory element that drives expression in the epidermis of the last three abdominal segments in females [8] (red bar in the blowup of region 1 in Figure 2A). This enhancer, which is the only identified sex-specific enhancer in the bab locus, is thought to control the sex-specific expression of both bab1 and bab2 [8]. This enhancer contains functionally characterized binding sites for the Abdominal-B (Abd-B) and doublesex (dsx) transcription factors [8]. None of the significantly associated polymorphisms affect these binding sites, and indeed there are no common polymorphisms in the 624 bp core enhancer. However, the variation surrounding this location is strongly associated with pigmentation variation (Figure 2A, 2B). Macroevolutionary changes in bab expression are largely due to sequence changes within the core enhancer but outside of the Abd-B and Dsx binding sites, indicating that CREs present large targets for functionally relevant mutations [8]. Our results reveal an even larger mutational target by demonstrating that sequence variation surrounding the core CRE can result in significant phenotypic variation without disrupting the primary function of the enhancer. This region also shows an excess of high-frequency derived alleles [10], suggesting that genetic variation in the region flanking the sex-specific abdominal CRE has been shaped by directional selection.

The second identified region is located in the intergenic region between bab1 and bab2 and contains a predicted and experimentally verified Polycomb response element (PRE) [12][13][14] (red bar in the blowup of region 2, Figure 2A). PREs regulate transcriptional activity over multiple cell generations by affecting chromatin state. As in the CRE region, the distribution of significantly associated polymorphisms is considerably wider than the functionally characterized PRE, with most effects located outside of the core region. These results suggest that PREs, as well as tissuespecific enhancers, present a large target for functionally and evolutionarily important mutations.

The third region showing a high concentration of significantly associated polymorphisms spans the promoter and transcription start site of bab2 (red bar in the blowup of region 3, Figure 2A). While promoters have been traditionally thought of as passive players in transcription, recent research suggests that promoters are often active in transcriptional regulation [15]. Several studies have shown that polymerases remain stalled on developmentally important genes, poised to begin transcription [16,17]. This pattern is observed on the bab2 promoter during embryonic development and in S2 cells [18], while the pupal stages are yet to be investigated. Our results suggest that not only are promoter regions involved in transcriptional regulation, but that sequence variation in these regions can have a significant impact on gene expression and phenotype. It is intriguing that no similar concentration of associated SNPs is observed around the bab1 promoter ( Figure 2A, 2B). We believe that the explanation for this discrepancy may lie in the differences between the transcriptional activity of bab1 and bab2 at the pupal stage (see below).

In addition to these three regions, there appears to be an excess of associated polymorphisms near the downstream exons of bab2. However, this observation can be attributed to cryptic population structure. The lines that comprise our sample were collected in two different years. The region of highly significant F ST between the two collections [10] coincides with the region of apparent association near the 39 exons of bab2 ( Figure 2B). When the samples from different years are examined separately, no association is observed in this region ( Figure 3A), suggesting that it contains no causative polymorphisms.

The existence of females with completely pigmented sixths abdominal segment distinguishes D. melanogaster from its close relatives D. simulans, D. mauritiana, and D. yakuba. To test whether derived SNP alleles are associated with darker phenotypes, we polarized DNA polymorphisms in the bab region using the genome of D. simulans. The 107 SNPs associated with phenotypic variation at FDR ,.2 ( Figure 3A) show a marginal bias towards derived alleles having a darker phenotype (53 of 76, 2-tailed Fisher's exact test P = 0.034; 31 polymorphisms not polarized).

Author Summary

Genes responsible for quantitative variation have been identified for a diverse range of phenotypes. However, much remains to be learned about the distribution of causative genetic variation within a locus. In this study, we investigated a locus that contributes to natural variation in abdominal pigmentation in Drosophila melanogaster. We found that the large phenotypic effect of this locus results from the cumulative action of many small-effect polymorphisms that are concentrated in three distinct functional regions: a promoter, a tissue-specific enhancer, and a Polycomb response element (a region involved in chromatin remodeling). The same regions influence the adult phenotype and transcript abundance, indicating that the causative sequence variants act by modulating transcription. Interestingly, these polymorphisms cluster near, but not within, the functionally validated regulatory regions, suggesting that DNA sequences surrounding core functional elements may play a key role in quantitative variation. Causative polymorphisms have small individual effect sizes

The effect size of each of these associated polymorphisms is small, but within each region, these polymorphisms form haplotypes that have a large effect on pigmentation. To examine these effects, we assigned each individual a haplotype score, which indicates the relative number of dark and light alleles they contain at the significant polymorphisms (see Material and Methods for details). A negative haplotype score indicates more light alleles than dark alleles, a positive score more dark than light and a score of zero indicates an equal number of dark and light alleles. There is a strong correlation between the haplotype score and pigmentation for all three regions ( Figure 4) (p,.001).

Within both regions 1 and 2 each polymorphism increases pigmentation by about 1% percent of the total variation (see Table 1). Some of these associated polymorphisms are likely not causative due to LD and false positives, but the effect size of each polymorphism is still small.

Despite each polymorphism only having a small effect on phenotype, together the polymorphisms result in haplotypes with large potential effects. Each region accounts for between 20% and 50% of the pigmentation variation. When we perform this analysis using haplotypes from all 3 regions, a similar pattern emerges; there is a small effect of each individual polymorphism, but a large overall effect (Table 1) (Figure 4). Together these three regions explain 84% of the pigmentation variation among the studied genotypes. The sum of regional effects appears to be greater than the total effect of polymorphisms in all three regions because our sample had a number of lines with completely pigmented A6 segments. Since pigmentation score cannot be higher than 100, such lines are saturated with dark alleles. In those haplotypes, each individual dark allele appears to have a smaller effect size.

Polymorphisms act in cis to influence transcript abundance

The location of associated polymorphisms in three non-coding regions suggests that QTL effects are due to differences in transcriptional regulation rather than protein function. Variation in gene expression has become a recent frontier in molecular and evolutionary genetics [19]. Local (cis-acting) regulatory variation may consist of expression quantitative trait nucleotides (eQTNs) in sequences responsible for controlling transcription. In yeast, for instance, transcriptional variation is frequently associated with eQTNs located close to promoter regions [20]. Other possible mechanisms include variation in splicing efficiency [21] and conformation [22]. A natural allele can incorporate multiple eQTNs with a complex interplay of these mechanisms [23], and cis-acting variants can also interact with distant (trans) regulatory factors in complex ways [24].

We examined cis-acting transcriptional variation in bab1 and bab2 directly using allele specific expression (ASE) [25,26]. We selected 51 of our lines that were all collected in a single year. Analysis of association between sequence variation and pigmentation in this sub-panel reveals the concentration of significant polymorphisms in the same three non-coding regions ( Figure 3A). We crossed each of these lines to two tester strains that contained low frequency SNPs in the coding regions of bab1 and bab2, allowing us to differentiate the two alleles. The use of two different tester strains allowed us to identify consistent cis effects and minimize complex interactions between cis and trans factors. Heterozygous female abdomens were collected at 48-56 hours after pupariation, when the bab genes are expressed in the abdominal epidermis [6]. In these heterozygotes, we measured the relative transcript levels of the bab1 and bab2 alleles from the line of interest and the tester strain, and corrected for differences in the tester strains (see Materials and Methods for details). In this experimental design, any differences in expression between the two parental alleles are due to cis-acting polymorphisms, and all 51 lines can be compared directly since they are measured relative to the same testers.

We used these transcription measurements to identify polymorphisms affecting transcription at the bab locus. We found a number of SNPs that are associated with variation in bab2 transcript abundance ( Figure 3B). Of the three previously identified regions affecting pigmentation, the regions flanking the PRE and the bab2 transcription start site contain polymorphisms with the strongest effects on bab2 transcript levels (p,.001; FDR ,.2). This overlap suggests that at least some of the variation in sex-specific pigmentation is the result of differential regulation of bab2 in different QTL alleles. We did not detect any effect of the region  straddling the sexually dimorphic abdominal CRE (region 1 in Figure 2A) on transcript abundance. This may be due to the fact that, in contrast to the PRE and the promoter, this CRE is active only in the epidermis of the last three abdominal segments, whereas the RNA samples used in the analysis of transcriptional variation comprised multiple other tissues where the bab genes are expressed, including the anterior abdominal segments, the ovaries, the oenocytes, muscles, and the genitalia. Alternatively, sequence variation in this CRE may influence transcription during a time period that was not sampled in our experiments. Although both bab1 and bab2 are expressed in the abdominal epidermis and contribute to the patterning of pigmentation [6,8], no sequence variants at the bab locus affect bab1 transcript levels (FDR ..5) (data not shown). Chromatin immunoprecipitation data show extensive binding of RNA polymerase II (polII) to the region surrounding the transcription start site of bab2, but not bab1, at the pupal stage [27]. In contrast, both promoter regions show similar polII binding during embryonic stages. Thus, it appears that sequence variation at the bab locus affects adult pigmentation primarily by modulating the transcription of bab2. It is possible, of course, that bab1 also contributes to phenotypic variation but at a different developmental stage that falls outside of the sampled period. Since pigmentation pattern is determined in a specific cell type at precise times during development [5,6,28], a more detailed understanding of how sequence variation affects transcription and translates into phenotypic differences will require quantitative analysis of gene expression with greater spatial and temporal resolution.

Previous research has identified two direct transcriptional regulators of bab, Abd-B and dsx [8]. Although the CREs responsible for bab expression in the abdominal epidermis are well defined [8], it is conceivable that additional regions modulate bab transcription in a redundant manner [29,30]. A search of the entire bab locus reveals 1412 potential Abd-B core binding sites (TTTAY) and 73 potential Dsx binding sites (ACWAWGT), which is higher than expected based on sequence composition (485 and 47 sites, respectively). However, none of the associated polymorphisms were found in potential Dsx binding sites, and only a few in potential Abd-B binding sites, in any of our analyses. This observation confirms that most variation in transcript levels and adult phenotype is due to sequence variation outside of known transcription factor binding sites.

Understanding the causative variation at the bab locus

The bab locus was identified as a large effect QTL responsible for most of the natural variation in female abdominal pigmentation [9]. By investigating the function of bab at two different levels, we were able to elucidate the fine structure of QTL alleles and characterize DNA sequence variation that affects transcription and phenotypic differences. Three major lessons emerge from this analysis. First, large-effect QTL alleles can reflect the cumulative effects of many nucleotide polymorphisms. This finding is in contrast to several cases where large QTLs are due to single coding or regulatory mutations with major phenotypic effects [31][32][33]. On the other hand, detailed analysis of a single gene responsible for 100% of a phenotypic difference between two Drosophila species revealed contributions from several independent regulatory regions [34]. Within Drosophila melanogaster, multiple polymorphisms in a single cis regulatory element of the ebony gene contribute to pigmentation differences between populations [35]. In addition many studies have shown the contribution of multiple polymorphisms to variation in protein function (e.g. [36][37][38][39]). These results suggest that accumulation of multiple mutations at the same locus may be a relatively common mechanism of microand macroevolutionary change.

Second, the causative polymorphisms are not distributed randomly throughout the locus, but are located in multiple, distinct functional regions. In the case of bab, the vast majority of polymorphisms associated with transcriptional and phenotypic variation are concentrated around three regions: the core promoter, tissue-specific enhancer, and Polycomb response element -the elements expected to contribute the most to transcriptional regulation. Thus, functional annotation of the human and model system genomes, such as that generated by the ENCODE and modENCODE projects [27,40], may go some way towards predicting the loci of evolutionary variation. Conversely, quantitative-genetic analyses of natural variation may help identify and annotate functional genomic regions.

The third major lesson is that the functional elements present larger targets than previously thought for phenotypically and evolutionarily important mutations. The regions detected in our analysis are much larger than the core elements defined previously by functional assays. Over 90% of significant polymorphisms are found around, but not precisely inside, the core CRE, PRE, and promoter. This pattern indicates that DNA sequence variation in regions surrounding the core elements has major phenotypic consequences. These sequence variants may act by modulating the regulatory activity of the functional elements, perhaps through subtle changes in chromatin structure or transcription factor recruitment, without disrupting their primary functions. As a result, important evolutionary changes may occur through mutations in DNA sequences that lack readily identifiable molecular functions. The rapidly growing body of genome-wide association studies will put this proposition to the test. However, the final lesson from the Drosophila bab locus is that fine-scale dissection of functional variation is only possible in genes with little linkage disequilibrium, underscoring the importance of model systems in framing the analysis of human genetic variation.

Materials and Methods

Lines selection and sequencing 96 inbred lines of D. melanogaster were chosen for sequencing the bab locus. 86 lines were chosen at random from the W1 and W3 inbred line collections maintained in the Nuzhdin lab. Progenitors of these lines were originally collected from Wolfskill Orchard in Winters, CA, and inbred by full sib mating for a minimum of 20 generations. 10 additional lines with low pigmentation scores were selected, including 8 lines from Winters and 2 from North Carolina. Sequencing was performed as described [10].

Pigmentation phenotyping and association mapping 3-5 day old females were scored for pigmentation as described [9]. All statistical tests were preformed using the R Statistical package (http://www.r-project.org/). Spearman's correlation test and associated p-values were calculated using ''cor.test'' command in the base Stats package for each polymorphism individually. False Discovery rates were calculated using the q-value package.

Identification of enriched regions and estimating effects size

To identify regions with an excess of associated points, we scanned the bab locus in 5 kb sliding windows, shifting the window in 500 bp increments. Regions where more than 0.12 of polymorphisms were significant were tentatively identified as enriched regions. We manually excluded the region that contained the cryptic population structure and then manually manipulated each remaining region to only include the significant points (i.e. we narrowed the region if the high score was the result of all of the polymorphisms being clustered on one side of the window).

To understand the effect size for each region, we created a region haplotype score for each individual. This haplotype score is the difference between the number of dark alleles and light alleles for significant polymorphisms divided by 2. ((Number of dark alleles -number of light alleles)/2). Thus an increase in the haplotype score by one indicates a change of a single light allele to a dark allele. A score of zero would indicate an equal number of dark and light alleles for that region. This formula was necessary because not all individuals had a genotype assigned for all polymorphism.

We then fit a linear model with the formula: pigmentation score = haplotype. The coefficient of the haplotype term is the average effect of each allele on the pigmentation score. To calculate the average effect size of the entire region we multiplied the average effect of each allele by the number of associated polymorphisms in that region.

Quantification of bab transcripts

We measured allele-specific expression of both bab1 and bab2 in 51 lines out of the 96 used for pigmentation mapping. Two lines that carried rare SNPs in the coding regions of bab1 and bab2 were chosen as the tester strains. Each of these tester strains was crossed to the 51 experimental lines in several replicates, creating F1 genotypes that were heterozygous for the tester and experimental alleles.

We collected 48-56 hour whole pupal abdomens from the female F1s. Individuals from replicate crosses were combined into pools of 10, and we attempted to obtain at least 2 pools from each cross. From each of these samples, a 218 bp fragment of bab1 and a 142 bp fragment of bab2, spanning the SNPs of interest, were amplified. We used Illumina high throughput sequencing of barcoded samples to measure the relative amounts of each allele as described [26]. To normalize the samples from the two tester lines, we took the residuals from a fixed cofactor ANOVA. We then mapped the expression variation across the bab region using the same procedures as for the adult phenotype.

Binding site predictions were made using custom R and Perl scripts for the sequences ACWAWGT (Dsx) and TTTAY (Abd-B).

Supporting Information

Figure 1 .
1Three representative females (lines W3-123, W3-112, and W3-135) showing the range of pigmentation variation. The arrow indicates A6, the segment analyzed. doi:10.1371/journal.pgen.1001275.g001

Figure 2 .
2Association mapping of female A6 pigmentation at the bab locus. (A) P-values from a Spearman rank correlation for each polymorphism identified in the bab locus. The dotted and solid red lines indicate FDR values of 0.3 and 0.4 respectively. 3 functional regions containing most associated polymorphisms (shaded blue rectangles) are shown in greater detail above. The sex-specific abdominal CRE (in region 1), Polycomb response element (region 2), and the bab2 promoter (region 3) are indicated by red bars. The yellow region contains an excess of linked polymorphisms, but is attributed to cryptic structure in our samples. (B) Black line shows the proportion of all polymorphisms in 5 kb sliding windows that are significantly associated with variation in pigmentation (FDR ,.4), plotted against the midpoint of the window. Overlayed in yellow is Fst between samples collected in separate years (see text for details). doi:10.1371/journal.pgen.1001275.g002

Figure 3 .
3Association mapping of adult pigmentation and transcriptional variation in the subset of lines used to measure bab expression. (A) Association between polymorphisms and pigmentation. (B) Association between polymorphisms and bab2 transcript levels. The p-values are from a Spearman rank correlation test for all polymorphisms. Regions shaded in blue are the same as in Figure 2. doi:10.1371/journal.pgen.1001275.g003

Figure 4 .
4Pigmentation and the haplotype of associated polymorphisms. The haplotype represents the relative number of dark and light alleles at the associated polymorphisms within the region. Individuals with negative scores have more light alleles than dark alleles and positive scores have more dark alleles than light alleles (see Materials and Methods for details). The line represents the best fit of the pigmentation as a function of haplotype score. Each of the three regions is displayed individually and all three together. doi:10.1371/journal.pgen.1001275.g004

Figure S1
S1Pairwise r2 LD values for significantly associated points in the 3 designated regions. Each square represents the pairwise r2 value. Darker squares indicate higher r2 values. Found at: doi:10.1371/journal.pgen.1001275.s001 (0.64 MB TIF)

Table 1 .
1Estimation of polymorphism and region effect size.Number of sites

Average effect size of
each polymorphism

Proportion of total
variation explained by
average polymorphism

Total estimated
effect of region

Proportion of total
variation explained

Region 1
55
0.80 (.19)
0.009
44
0.512

Region 2
21
0.97 (.28)
0.011
20.37
0.237

Region 3
29
1.02 (.30)
0.012
29.58
0.344

All 3 regions
105
0.69 (.10)
0.008
72.45
0.842

doi:10.1371/journal.pgen.1001275.t001

PLoS Genetics | www.plosgenetics.org
January 2011 | Volume 7 | Issue 1 | e1001275
AcknowledgmentsWe thank the Joint Genome Institute community sequencing program, Wendy S. Schackwitz, and Len A. Pennacchio for help with the sequencing. We thank Jennifer Brisson and three anonymous reviewers for helpful comments on the manuscript.Author ContributionsConceived and designed the experiments: RDB AK SVN. Performed the experiments: RDB. Analyzed the data: RDB AK SVN. Wrote the paper: RDB AK SVN.
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