Quantitative trait loci mapping of dauer larvae development in growing populations of <i>Caenorhabditis briggsae</i>

James Green; Simon Harvey

doi:10.12688/f1000research.7546.1

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Research Note

Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae

[version 1; peer review: 3 approved with reservations]

James Green¹, Simon Harvey¹

PUBLISHED 15 Dec 2015

Author details Author details

¹ Biomolecular Research Group, School of Human and Life Sciences, Canterbury Christ Church University, Canterbury, UK

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REVIEWER STATUS

Abstract

The life cycles of many free-living nematodes contain developmental switches that allow individuals to either develop directly to adulthood, or to arrest development as a stress resistant and long-lived dauer larval stage. Here, in a panel of Caenorhabditis briggsae recombinant inbred lines derived from the isolates HK104 x AF16, we use methodologies developed for C.elegans to map quantitative trait loci (QTLs) affecting the number of dauer larvae present at the point of food patch exhaustion. These analyses provide strong support for three QTLs and are suggestive of a further two.

Keywords

dauer larvae, Caenorhabditis briggsae, QTL, nematode, natural variation

Corresponding author: Simon Harvey

Competing interests: No competing interests were disclosed.

Grant information: The author(s) declared that no grants were involved in supporting this work.

Copyright: © 2015 Green J and Harvey S. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

How to cite: Green J and Harvey S. Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae [version 1; peer review: 3 approved with reservations]. F1000Research 2015, 4:1447 (https://doi.org/10.12688/f1000research.7546.1) First published: 15 Dec 2015, 4:1447 (https://doi.org/10.12688/f1000research.7546.1) Latest published: 15 Dec 2015, 4:1447 (https://doi.org/10.12688/f1000research.7546.1)

Introduction

Caenorhabditis elegans is an important model species, but there is a need to further understand its ecology and that of the other species with which it is associated^1–3. Active, growing, populations of Caenorhabditis nematodes are normally associated with nutrient and bacteria-rich substrates such as rotten fruit or very fresh compost^1,3,4. Growing populations can reach very large sizes, and worms would be expected to have optimized fitness (population growth and/or the production of dispersal stages) under such conditions. Appropriate development of growing worms as dauer larvae, the developmentally-arrested alternate third larval stage, is therefore likely to be central to genotype survival.

We have previously looked at dauer larvae development in growing populations of C. elegans^5–7. These analyses identified both extensive variation between wild isolates in the number of dauer larvae present when a bacterial food patch is exhausted⁶ and large numbers of QTLs effecting this trait in introgression lines (ILs) produced from the isolates N2 and CB4856^6,7. Naturally, C. elegans has however been found to be associated with both C. remanei and C. briggsae^1,3,8. Here, using the methodologies developed for C. elegans, we have investigated dauer larvae formation in growing populations in a panel of C. briggsae recombinant inbred lines (RILs) produced from the isolates AF16 and HK104⁹.

Methods

The C. briggsae RILs were generated from reciprocal crosses between males and sperm-depleted hermaphrodites of isolates HK104 and AF16⁹ and were obtained from Asher Cutter (University of Toronto). Worms were maintained using standard methods¹⁰ and all assays were conducted at 20°C. Assays were performed as previously described⁶, with populations initiated from single fourth larval stage worms (L4s) grown from synchronized, arrested, L1s with 100 μl of a 20% w/v suspension of Escherichia coli in water and monitored daily until food exhaustion. At this point, the population size and the number of dauer larvae were determined^5,6. RILs were analyzed in three experimental blocks with AF16 and HK104 assayed in each block and five populations initiated per genotype. Treatments (genotypes) were randomized within blocks and plates were blind coded such that counts were performed without knowledge of worm genotype. Populations that failed to grow were discarded.

The RILs have previously been genotyped at 451 markers distributed across all six chromosome pairs⁹ and these data were used for this analysis. QTL mapping was performed by composite interval mapping (CIM) using QTL Cartographer v2.5¹¹. Genome-wide thresholds (0.05) were estimated based on 1000 permutations of the data and CIM analysis undertaken with a 1.0cM walk speed.

Results

Block	Line	Pop	Dauer
1	PB1103	77000	0
1	PB1103	30000	0
1	PB1103	29000	320
1	PB1103	37000	40
1	PB1104	72000	20
1	PB1104	46000	0
1	PB1104	58000	110
1	PB1104	55000	10
1	PB1104	59000	0
1	PB1105	63000	0
1	PB1105	63000	0
1	PB1105	39000	0
1	PB1105	114000	0
1	PB1105	37000	0
2	PB1106	34000	870
2	PB1106	53000	200
2	PB1106	47000	1350
2	PB1106	61000	830
2	PB1106	52000	270
2	PB1107	25000	50
2	PB1107	45000	120
2	PB1107	78000	60
2	PB1107	55000	20
2	PB1107	58000	0
3	PB1109	36800	201
3	PB1109	40000	319
3	PB1109	37600	205
3	PB1109	67200	200
3	PB1109	31200	263
1	PB1110	54000	0
1	PB1110	61000	0
1	PB1110	60000	0
1	PB1110	67000	0
1	PB1110	73000	10
1	PB1111	18000	30
1	PB1111	35000	80
1	PB1111	41000	90
1	PB1111	34000	0
1	PB1111	45000	140
1	PB1112		70
1	PB1112	38000	0
1	PB1112	30000	170
1	PB1112	46000	0
1	PB1112	68000	0
2	PB1113	45000	860
2	PB1113	27000	650
2	PB1113	39000	810
2	PB1113	35000	850
2	PB1113	39000	770
1	PB1115	29000	30
1	PB1115	66000	120
1	PB1115	42000	0
1	PB1115	40000	40
1	PB1116	41000	970
1	PB1116	53000	20
1	PB1116	16000	800
1	PB1116	21000	180
1	PB1117	46000	1250
1	PB1117	23000	1310
1	PB1117	46000	750
1	PB1117	42000	1020
1	PB1117	70000	60
1	PB1118	32000	0
1	PB1118	54000	20
1	PB1118	61000	20
1	PB1118	54000	10
1	PB1118	32000	0
1	PB1119	27000	1350
1	PB1119	21000	360
1	PB1119	31000	840
1	PB1119	40000	1050
3	PB1121	24000	37
3	PB1121	19200	46
1	PB1121	63000	0
1	PB1121	62000
1	PB1121	68000	610
1	PB1121	86000	1010
1	PB1121	69000	20
2	PB1125	45000	640
2	PB1125	50000	60
2	PB1125	25000	690
2	PB1125	57000	450
2	PB1125	58000	1030
1	PB1126	54000	240
1	PB1126	62000	720
1	PB1126	45000	800
1	PB1126	53000	840
1	PB1126	63000	820
1	PB1127	16000	620
1	PB1127	61000	290
1	PB1127	35000	50
1	PB1127	54000	150
1	PB1128	7000	310
1	PB1128	17000	380
1	PB1128	24000	410
1	PB1128	38000	180
2	PB1129	29000	320
2	PB1129	36000	300
2	PB1129	42000	170
2	PB1129	64000	690
2	PB1129	49000	490
2	PB1130	42000	980
2	PB1130	51000	700
2	PB1130	37000	420
2	PB1130	35000	230
2	PB1130	32000	70
2	PB1132	36000	560
2	PB1132	52000	770
2	PB1132	41000	350
2	PB1132	45000	840
2	PB1132	47000	710
2	PB1133	44000	100
2	PB1133	34000	340
2	PB1133	21000	380
2	PB1133	54000	100
2	PB1133	37000	150
1	PB1133	26000	0
1	PB1134	42000	70
1	PB1134	43000	900
1	PB1134	51000	730
1	PB1134	54000	1070
1	PB1134	35000	490
1	PB1135	60000	50
1	PB1135	44000	0
1	PB1135	39000	10
1	PB1135	56000	0
1	PB1135	52000	0
1	PB1136	12000	330
1	PB1136	47000	560
1	PB1136	32000	210
1	PB1136	48000	1040
1	PB1136	40000	360
2	PB1137	43000	210
2	PB1137	55000	190
2	PB1137	27000	250
2	PB1137	34000	290
2	PB1137	27000	170
2	PB1138	41000	320
2	PB1138	58000	30
2	PB1138	45000	60
2	PB1138	90000	100
1	PB1139	35000	870
1	PB1139	48000	660
1	PB1139	67000	120
1	PB1139	18000	20
1	PB1139	48000	0
1	PB1140	59000	310
1	PB1140	53000	620
1	PB1140	35000	550
1	PB1140	42000	910
1	PB1140	25000	470
1	PB1141	50000	720
1	PB1141	71000	60
1	PB1141	77000
1	PB1141	27000	20
1	PB1141	34000	1040
2	PB1142	35000	340
2	PB1142	65000	680
2	PB1142	52000	1260
2	PB1142	52000	1350
1	PB1143	55000	0
1	PB1143	52000	260
1	PB1143	57000	10
1	PB1143	58000	960
2	PB1144	49000	1010
2	PB1144	53000	830
1	PB1145	42000	110
1	PB1145	41000	240
1	PB1145	30000	0
1	PB1145	23000	0
1	PB1145	31000	370
1	PB1146	25000	770
1	PB1146	32000	470
1	PB1146	23000	570
1	PB1146	139000	760
1	PB1146	39000	0
1	PB1147	31000	80
1	PB1147	9000	130
1	PB1147	38000	150
1	PB1147	55000	270
1	PB1147	54000	370
2	PB1148	43000	40
2	PB1148	31000	100
2	PB1148	33000	110
1	PB1149	90000	0
1	PB1149	24000	0
1	PB1149	93000	40
1	PB1149	27000	0
1	PB1149	33000	0
2	PB1151	44000	710
2	PB1151	35000	690
2	PB1151	49000	510
2	PB1151	47000	390
1	PB1152	72000	260
1	PB1152	105000	700
1	PB1152	23000	10
1	PB1152	70000	230
1	PB1152	63000	890
2	PB1153	42000	210
This is a portion of the data; to view all the data, please download the file.

Dataset 1.The number of dauer larvae and the population size at food exhaustion for replicate populations of the RILs and the respective AF16 and HK104 controls from three experimental blocks.

The AF16 and HK104 data are presented in Figure 1. The RIL data are used to calculate the trait values used in QTL mapping.

0.006004	0.012008	0.024109	0.030113	0.042214	0.054316	0.06032	0.084843	0.103117	0.194853	0.213127	0.290679	0.30278	0.327304	0.448676	0.479529	0.49163	0.516154	0.522158	0.528162	0.585163	0.603437	0.63429	0.640294	0.658568	0.683092	0.689096	0.701197	0.707201	0.713205	0.731479	0.737483	0.743486	0.755588	0.761592	0.767596	0.779697	0.785701	0.797802	0.828655	0.834659	0.878415	0.890516	0.91504	0.952303	0.976827	1.027161	1.051685	1.063787	1.075888	1.119644	1.131745	1.137749	1.194751	1.251752	1.343488	1.349492	1.380345	1.444103	1.543085	1.59342	1.624272	1.668028	1.698881	1.717155
0.006004	0.012008	0.018012	0.124347	0.130351	0.142452	0.166976	0.179077	0.203601	0.247357	0.265631	0.322632	0.340906	0.371759	0.402612	0.408615	0.426889	0.432893	0.438897	0.46975	0.513506	0.53803	0.550131	0.562233	0.586756	0.59276	0.598764	0.617038	0.635312	0.653586	0.65959	0.665594	0.671598	0.677602	0.714864	0.720868	0.73297	0.738974	0.751075	0.775599	0.7877	0.793704	0.805805	0.811809	0.817813	0.829914	0.835918	0.89292	0.949921	1.071294	1.083395	1.101669	1.138932	1.163455	1.169459	1.181561	1.231896	1.262748	1.281022	1.287026	1.393361	1.405462	1.436315	1.442319	1.45442	1.466522	1.53028	1.548553	1.560655	1.578929	1.584933	1.590937	1.609211	1.615215	1.621219	1.63332	1.639324	1.645328
0.006004	0.018105	0.030207	0.05473	0.079254	0.085258	0.091262	0.109536	0.166537	0.178639	0.203162	0.221436	0.22744	0.312034	0.330308	0.387309	0.405583	0.47619	0.494464	0.500468	0.512569	0.56957	0.600423	0.612524	0.649787	0.68064	0.686644	0.723907	0.736008	0.748109	0.754113	0.760117	0.766121	0.772125	0.778129	0.784133	0.802407	0.820681	0.838955	0.844958	0.863232	0.869236	0.881338	0.887342	0.911865	0.917869	0.923873	0.942147	0.960421	0.991274	0.997278	1.009379	1.015383	1.033657	1.045758	1.10276	1.127283	1.133287	1.151561	1.157565	1.163569	1.169573	1.200426	1.237689	1.24979	1.255794	1.274068	1.304921	1.329445	1.393202	1.417726	1.429828	1.480163	1.486166
0.006004	0.04976	0.055764	0.106099	0.130623	0.167885	0.205148	0.211152	0.242005	0.254106	0.284959	0.34196	0.354062	0.360065	0.378339	0.384343	0.4281	0.481757	0.496935	0.527787	0.55864	0.570741	0.582843	0.620106	0.626109	0.644383	0.650387	0.668661	0.674665	0.680669	0.686673	0.698774	0.710876	0.716879	0.728981	0.734985	0.759508	0.77161	0.789884	0.795888	0.801892	0.826415	0.844689	0.862963	0.875065	0.893339	0.917862	0.942386	0.94839	0.954394	0.960398	0.966402	0.978503	1.003027	1.009031	1.015035	1.021039	1.03314	1.045241	1.076094	1.082098	1.119361	1.156624	1.168725	1.180826	1.224582	1.236684	1.242688	1.24567	1.32673	1.332734	1.338738	1.369591	1.433349	1.470612	1.482713	1.488717	1.559324	1.565328	1.59618	1.614454	1.626556	1.64483	1.650834	1.656838	1.668939	1.732697
0.030853	0.036857	0.048958	0.054962	0.060966	0.07924	0.085244	0.109768	0.14062	0.146624	0.164898	0.176999	0.195273	0.201277	0.219551	0.225555	0.231559	0.249833	0.268107	0.274111	0.311374	0.329648	0.347922	0.366196	0.3722	0.403053	0.421326	0.478328	0.484332	0.496433	0.527286	0.558138	0.61514	0.633414	0.639418	0.657692	0.663696	0.669699	0.675703	0.681707	0.687711	0.705985	0.730509	0.736513	0.742517	0.748521	0.766795	0.772799	0.791072	0.815596	0.827698	0.845972	0.864245	0.882519	0.888523	0.900625	0.931477	0.96233	0.986854	1.005128	1.19163	1.203731	1.222005	1.240279	1.264803	1.276904	1.289005	1.313529	1.32563	1.337732	1.343736	1.421287	1.445811	1.451815	1.536408	1.548509	1.560611	1.572712	1.597236	1.62176	1.646284	1.652287	1.658291	1.664295	1.670299	1.682401	1.694502	1.706603

Dataset 2.Marker distances (from Ross et al., 2011) for informative markers in the RILs analysed.

data	cb34755	cb9210	cb9192	cb6850	cb62971	cb62993	cbv25636	cb2044	cb61995	cb45568	cb45465	cb8644	cb15282	cb15284	cbv29577	cb66726	cb66765	cb66841	cb66970	cb57701	cbv18421	cb57379	cb1410	cb44525	cb44577	cb44346	cb16165	cb16121	cb16081	cb51634	cb38955	cb21981	cb66225	cb14869	cb28302	cb54162	cb54171	cb54270	cb54288	cb54371	cb54417	cb54442	cb25626	cb63977	cb19771	cb37388	cb19687	cb19655	cb55599	cb55695	cb62188	cb3139	cb64078	cbv26864	cb64120	cb36822	cb69435	cb58901	cb58858	cbv20005	cb57857	cb44738	cb23738	cb23809	cbv25999	cb987	cb41559	cb41422	cb39492	cb41334	cb18765	cb62203	cb61868	cb70341	cb16372	cb66334	cb44149	cb44178	cb55400	cb55366	cb55256	cb55205	cb55201	cbv16403	cb55101	cb13018	cb41016	cb6450	cb51910	cb3372	cb3404	cb16739	cb16720	cbv13858	cb52181	cb4596	cb54826	cb4210	cb23118
ril3_PB1105	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1106	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B
ril3_PB1107	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1112	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1113	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1115	A	A	A	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1116	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1117	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B
ril3_PB1118	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1119	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1121	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1125	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1126	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1127	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A
ril3_PB1128	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1129	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A
ril3_PB1130	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1132	B	B	B	B	B	B	B	B	B	B	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1133	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1134	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	A	A	A	A	A	A	A	A	A	A
ril3_PB1135	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1136	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A
ril3_PB1137	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1138	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1139	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1140	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1141	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1142	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1143	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1144	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1145	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	B	B	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1146	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1147	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1148	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1149	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1151	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1153	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1154	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B
ril3_PB1155	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1156	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B
ril3_PB1157	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B
ril3_PB1158	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1159	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1162	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1163	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1164	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1165	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1166	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1167	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1168	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1169	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1170	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1171	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B
ril3_PB1172	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1173	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B
ril3_PB1174	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1177	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1178	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril3_PB1179	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1182	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A
ril3_PB1185	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	A	A	A	A	A	A	A	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1187	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1189	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A
ril3_PB1191	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1192	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril3_PB1193	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1206	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1214	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1215	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1217	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	B	B	B
ril4_PB1218	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1219	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1220	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1222	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1231	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1232	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	A	A	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1233	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1235	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1240	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1241	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B
ril4_PB1243	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A
ril4_PB1245	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	A	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B	B
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This is a portion of the data; to view all the data, please download the file.

Dataset 3.RIL genotypes (from Ross et al., 2011) and analysed trait values for the RILs.

In combination with the marker distances, these data were used for QTL mapping and the generation of the results shown in Figure 2 and Table 1.

Table 1. QTLs affecting the number of dauer larvae at food exhaustion.

Position is the 1 LOD interval for the QTL. R² denotes the proportion of the inter-RIL variance explained by the QTL, and effect indicates how the QTL alters dauer larvae numbers, with a positive value indicating that the AF16 allele increases the number of dauer larvae compared to the HK104 allele.

Chromosome	QTL	Position (Mbp)	R²	Effect	Support
II	1	13.7–14.5	8.3	-	AF16-scaled
IV	2	14.9–17.5	12.6	-	AF16-scaled
		15.1–16.4	12.6	-	HK104-scaled
		15.1–15.6	14.4	-	block 2
		14.9–16.4	37.5	-	block 3
V	3	2.5–5.6	8.0	+	AF16-scaled
		3.0–5.6	8.4	+	HK104-scaled
		2.8–14.3	20.0	+	block 2
V	4	16.1–16.8	17.4	-	block 3
X	5	3.0–6.2	8.8	+	AF16-scaled
		0–11.6	15.5	+	HK104-scaled
		0–7.6	17.2	+	block 1
		4.7–6.2	26.3	+	block 3

Comparing the AF16 and HK104 controls between the experimental blocks indicates that the number of dauer larvae at food exhaustion varies extensively between blocks (H = 11.06, p = 0.004 and H = 8.21, p = 0.007, for AF16 and HK104, respectively: Figure 1A). This variation is much greater than that seen for population size at food exhaustion (Figure 1B). Such variability between experimental blocks has been observed before with this assay, and is likely to be due to variation in either the actual or perceived food quality between batches of bacteria⁶.

Given the variation in dauer larvae formation between the experimental blocks we considered it unlikely that analysis of raw data would identify any QTLs, and this is what was observed (Figure 2A). Two approaches were therefore taken to determine if we could control for this variation. Firstly, we scaled the number of dauer larvae observed for each RIL either to the AF16 or HL104 controls within that experimental block (Figure 2A), e.g. the AF16 scaled value for a RIL was determined as (RIL mean - AF16 mean)/AF16 mean and this was used for mapping. Secondly, we analyzed each of the experimental blocks independently (Figure 2B).

Figure 1. Dauer larvae formation is variable across experimental blocks.

The A) number of dauer larvae, and B) population size, at food exhaustion in AF16 and HK104 controls in the three experimental blocks. Values from each plate are shown, with red bars indicating the median values.

Figure 2. Quantitative trait mapping identifies regions affecting the number of dauer larvae at food exhaustion.

A) CIM of the number of dauer larvae (solid line), and the number of dauer larvae scaled to the numbers in the AF16 (dashed line) or HK104 (dotted line) controls from that experimental block. B) CIM of the number of dauer larvae observed in each experimental block separately. Horizontal lines indicate thresholds.

In combination, these approaches identify three QTLs on chromosomes IV, V and the X, that affect the number of dauer larvae present at food exhaustion (co-localizing QTLs present in both Figure 2A and B) (Table 1). The analysis is also suggestive of additional QTLs on chromosomes II and V (Figure 2A and B; Table 1). A similar analysis of the population size at food exhaustion did not detect any QTLs.

Discussion

For a comparative analysis of dauer larvae development in growing populations, a panel of C. briggsae RILs were analyzed. These analyses indicate that the general properties of growing populations of C. briggsae are similar to those described for C. elegans^5,6. This means these methodologies will be suitable for more direct comparisons between the species. The RIL analysis identifies a number of QTLs (Figure 2; Table 1) although these are not resolved to narrow genomic regions and therefore contain many hundreds of genes. They do however provide a starting point for attempts to identify causal loci.

Data availability

F1000Research: Dataset 1. The number of dauer larvae and the population size at food exhaustion for replicate populations of the RILs and the respective AF16 and HK104 controls from three experimental blocks, 10.5256/f1000research.7546.d109088¹²

F1000Research: Dataset 2. Marker distances (from Ross et al., 2011) for informative markers in the RILs analysed, 10.5256/f1000research.7546.d109089¹³

F1000Research: Dataset 3. RIL genotypes (from Ross et al., 2011) and analysed trait values for the RILs, 10.5256/f1000research.7546.d109090¹⁴

Author contributions

JG and SH conceived the study and designed the experiments. JG carried out the research. SH and JG analyzed the data, wrote the paper and have agreed to the final content.

Competing interests

No competing interests were disclosed.

Grant information

The author(s) declared that no grants were involved in supporting this work.

Acknowledgements

We thank Asher Cutter for the C. briggsae RILs. We thank Helen Orbidans for her assistance with the assays.

Faculty Opinions recommended

References

1. Félix MA, Braendle C: The natural history of Caenorhabditis elegans. Curr Biol. 2010; 20(22): R965–R969. PubMed Abstract | Publisher Full Text
2. Petersen C, Dirksen P, Schulenburg H: Why we need more ecology for genetic models such as C. elegans. Trends Genet. 2015; 31(3): 120–127. PubMed Abstract | Publisher Full Text
3. Frézal L, Félix MA: C. elegans outside the Petri dish. eLife. 2015; 4: e05849. PubMed Abstract | Publisher Full Text | Free Full Text
4. Kiontke KC, Félix MA, Ailion M, et al.: A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits. BMC Evol Biol. 2011; 11(1): 339. PubMed Abstract | Publisher Full Text | Free Full Text
5. Green JW, Harvey SC: Development of Caenorhabditis elegans dauer larvae in growing populations. Nematology. 2012; 14(2): 165–173. Publisher Full Text
6. Green JW, Snoek LB, Kammenga JE, et al.: Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans. Heredity (Edinb). 2013; 111(4): 306–313. PubMed Abstract | Publisher Full Text | Free Full Text
7. Green JW, Stastna JJ, Orbidans HE, et al.: Highly polygenic variation in environmental perception determines dauer larvae formation in growing populations of Caenorhabditis elegans. PLoS One. 2014; 9(11): e112830. PubMed Abstract | Publisher Full Text | Free Full Text
8. Félix MA, Duveau F: Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae. BMC Biol. 2012; 10(1): 59. PubMed Abstract | Publisher Full Text | Free Full Text
9. Ross JA, Koboldt DC, Staisch JE, et al.: Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination. PLoS Genet. 2011; 7(7): e1002174. PubMed Abstract | Publisher Full Text | Free Full Text
10. Stiernagle T: Maintenance of C. elegans. (february 11, 2006), WormBook. ed. the C. elegans Research Community, WormBook, 2006. PubMed Abstract | Publisher Full Text
11. Wang SC, Basten CJ, Zeng ZB: Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC, 2007. Reference Source
12. Green J, Harvey S: Dataset 1 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015a. Data Source
13. Green J, Harvey S: Dataset 2 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015b. Data Source
14. Green J, Harvey S: Dataset 3 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015c. Data Source

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¹ Biomolecular Research Group, School of Human and Life Sciences, Canterbury Christ Church University, Canterbury, UK

Competing interests

No competing interests were disclosed.

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The author(s) declared that no grants were involved in supporting this work.

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Green J and Harvey S. Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae [version 1; peer review: 3 approved with reservations]. F1000Research 2015, 4:1447 (https://doi.org/10.12688/f1000research.7546.1)

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Reviewer Report 01 Feb 2016

Christian Braendle, Institute of Biology Valrose, CNRS UMR7277, University of Nice Sophia Antipolis, Nice, France

Approved with Reservations

https://doi.org/10.5256/f1000research.8126.r11575

This research note presents a very brief summary of a QTL analysis based on a phenotyping assay of dauer formation (at food exhaustion) in C. briggsae AI-RILs.

The main issue here is the low reproducibility of the phenotyping assay across replicates ... Continue reading

This research note presents a very brief summary of a QTL analysis based on a phenotyping assay of dauer formation (at food exhaustion) in C. briggsae AI-RILs.

The main issue here is the low reproducibility of the phenotyping assay across replicates and blocks. Given this problem, it appears rather surprising that some significant QTL were picked up. Further assays would be required to validate these, and this might be worthwhile doing (given that reproducible assays can be developed).

If I understood correctly, adjusted or unadjusted numbers of dauers were used as trait values – why not rather use the proportion of dauers (or number of dauers adjusted by population size) for mapping? [higher densities are expected to increase dauer formation]

What is the relationship between proportion of dauers and population size? Can the variability in dauer formation be explained by variability in population size? Does mapping of population size give the same QTLs?

Figure 1 shows how strong replicate and block effects are, just by looking at the parental lines, AF16 and HK104. Does an ANOVA analysis of these data (or the RIL data) actually reveal a significant genotype effect (so far, only block effects are given)? What are the heritabilities?

How does this QTL analysis in briggsae compare to the QTL analysis done in elegans (N2xCB4856) – number of QTLs, regions?

In summary: it would be useful to add some more info and data analysis to this note.

Additional comments

perhaps mention that the lines used are advanced-intercross recombinant inbred lines (AI-RILs)
the authors mention 451 markers but I had the impression many more were generated in Ross et al. 2011

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 20 Jan 2016

Eric Haag, Department of Biology, University of Maryland, College Park, MD, USA

Approved with Reservations

https://doi.org/10.5256/f1000research.8126.r11992

In this report, Green and Harvey address the genetic architecture governing variation in one of the best-studied examples of developmental plasticity in animas, the dauer larva Caneorhabditis nematodes.

Dauer larvae are dispersive forms of nearly all terrestrial nematodes, and are typically ... Continue reading

In this report, Green and Harvey address the genetic architecture governing variation in one of the best-studied examples of developmental plasticity in animas, the dauer larva Caneorhabditis nematodes.

Dauer larvae are dispersive forms of nearly all terrestrial nematodes, and are typically induced by the onset of food limitation and/or pheromone cues that are correlated with it. Given that the correctness of the decision to form a dauer (and thus abandon the direct path to reproductive adulthood) is expected to be highly contingent upon the exact ecological context in which it occurs, this trait should be both subject to strong selection and be potentially variable. This may be especially true for C. briggsae, which has a greater degree of geographic population structure than does its more famous congener, C. elegans.

The authors take advantage of a set recombinant inbred lines between genetically distinct strains (lines that my own lab played a role in characterizing in 2011; see the Ross et al. reference) to examine whether there is indeed heritable variation for dauer formation, and if so whether there are any loci of variation with effect size big enough to show significant association with dauer formation. I have no doubts that this project is addressing a significant issue in biology, and the authors make some headway.

General enthusiasm aside, as Erik Andersen also notes in his review the main concern here is the noisiness of the assay that is mapped. In understanding how the dauer formation assays is done, I think I may see why it is so noisy. The authors are founding each plate with a single L4 larva and a fixed amount of food. This is easy to reproduce, so I see the appeal. However, to then reach starvation on the plate, we presumably are looking at the grandchildren of the founder worm. As an exponential process, tiny differences in the generation time or rate of egg laying in the first few hours would produce big variability in the total number of worms, and also in the increase of the signals that are stimulatory for dauer production.

One could try to model the nature of this variation, but I don’t think this is author’s goal. An alternative, though admittedly requiring a whole new set of experiments, would be to seed plates with 20 or 200 L4 larvae, and score dauer formation in the first generation. This ought to be far less variable, yet should also be assessing the same basic phenotype, which could be seen as the threshold signal required to induce dauer development. Rather than repeating the entire scan, perhaps the authors could take their most discordant RILs and repeat this assay on them? It may produce a stronger signal, or at least confirm the initial screen.

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I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 18 Jan 2016

Erik C. Andersen, Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA

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https://doi.org/10.5256/f1000research.8126.r11792

Using techniques published previously, the authors measure the number of dauer larvae present at food patch exhaustion for a collection of Caenorhabditis briggsae recombinant lines. The dauer phenotype results are very noisy (as observed previously), but after data transformation quantitative trait ... Continue reading

Using techniques published previously, the authors measure the number of dauer larvae present at food patch exhaustion for a collection of Caenorhabditis briggsae recombinant lines. The dauer phenotype results are very noisy (as observed previously), but after data transformation quantitative trait loci (QTL) are detected using composite interval mapping. These results suggest that some regions of the C. briggsae genome confer differences in dauer formation, but more analysis needs to be performed to know more definitively. I would soften the abstract language (e.g. "strong support") to reflect the uncertainty in the results.

Given the variability of the phenotype measurements and the small number of recombinant lines, I am surprised about the QTL results. I would have expected no QTL to be detected, as observed in the untransformed data. It would be nice to see some more analyses, including the following:

What is the broad-sense heritability of this trait for these strains? It would be good to know how much of the trait variance you might expect to map to genomic regions in this study.
What are the results of a non-parametric linkage mapping? Composite interval mapping has many caveats. Are these QTL robust to mapping technique?
The significance threshold seems low given the allele frequency skews present in this mapping population. Please explain more about how that threshold was determined and how the skews were controlled.
Can you plot the RIL phenotypes before and after transformation so we can assess how well the transformation affected the high variability of the assay?

If the detected QTL represent expected amounts of the genetic variance, are robust to mapping technique, and are significant with respect to the known allele frequency skews in this mapping population, then I believe this article offers preliminary evidence that some genomic regions confer differences in dauer larvae formation. These results suggest genomic regions to test for causality in the trait of interest, including regions that could lead to conserved interspecific variation between C. elegans and C. briggsae.

Competing Interests: No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Erik C. Andersen, Northwestern University, Evanston, USA
Eric Haag, University of Maryland, College Park, USA
Christian Braendle, University of Nice Sophia Antipolis, Nice, France

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01 Feb 2016 | for Version 1

Christian Braendle, Institute of Biology Valrose, CNRS UMR7277, University of Nice Sophia Antipolis, Nice, France

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This research note presents a very brief summary of a QTL analysis based on a phenotyping assay of dauer formation (at food exhaustion) in C. briggsae AI-RILs.

The main issue here is the low reproducibility of the phenotyping assay across replicates and blocks. Given this problem, it appears rather surprising that some significant QTL were picked up. Further assays would be required to validate these, and this might be worthwhile doing (given that reproducible assays can be developed).

If I understood correctly, adjusted or unadjusted numbers of dauers were used as trait values – why not rather use the proportion of dauers (or number of dauers adjusted by population size) for mapping? [higher densities are expected to increase dauer formation]

What is the relationship between proportion of dauers and population size? Can the variability in dauer formation be explained by variability in population size? Does mapping of population size give the same QTLs?

Figure 1 shows how strong replicate and block effects are, just by looking at the parental lines, AF16 and HK104. Does an ANOVA analysis of these data (or the RIL data) actually reveal a significant genotype effect (so far, only block effects are given)? What are the heritabilities?

How does this QTL analysis in briggsae compare to the QTL analysis done in elegans (N2xCB4856) – number of QTLs, regions?

In summary: it would be useful to add some more info and data analysis to this note.

Additional comments

perhaps mention that the lines used are advanced-intercross recombinant inbred lines (AI-RILs)
the authors mention 451 markers but I had the impression many more were generated in Ross et al. 2011

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20 Jan 2016 | for Version 1

Eric Haag, Department of Biology, University of Maryland, College Park, MD, USA

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In this report, Green and Harvey address the genetic architecture governing variation in one of the best-studied examples of developmental plasticity in animas, the dauer larva Caneorhabditis nematodes.

Dauer larvae are dispersive forms of nearly all terrestrial nematodes, and are typically induced by the onset of food limitation and/or pheromone cues that are correlated with it. Given that the correctness of the decision to form a dauer (and thus abandon the direct path to reproductive adulthood) is expected to be highly contingent upon the exact ecological context in which it occurs, this trait should be both subject to strong selection and be potentially variable. This may be especially true for C. briggsae, which has a greater degree of geographic population structure than does its more famous congener, C. elegans.

The authors take advantage of a set recombinant inbred lines between genetically distinct strains (lines that my own lab played a role in characterizing in 2011; see the Ross et al. reference) to examine whether there is indeed heritable variation for dauer formation, and if so whether there are any loci of variation with effect size big enough to show significant association with dauer formation. I have no doubts that this project is addressing a significant issue in biology, and the authors make some headway.

General enthusiasm aside, as Erik Andersen also notes in his review the main concern here is the noisiness of the assay that is mapped. In understanding how the dauer formation assays is done, I think I may see why it is so noisy. The authors are founding each plate with a single L4 larva and a fixed amount of food. This is easy to reproduce, so I see the appeal. However, to then reach starvation on the plate, we presumably are looking at the grandchildren of the founder worm. As an exponential process, tiny differences in the generation time or rate of egg laying in the first few hours would produce big variability in the total number of worms, and also in the increase of the signals that are stimulatory for dauer production.

One could try to model the nature of this variation, but I don’t think this is author’s goal. An alternative, though admittedly requiring a whole new set of experiments, would be to seed plates with 20 or 200 L4 larvae, and score dauer formation in the first generation. This ought to be far less variable, yet should also be assessing the same basic phenotype, which could be seen as the threshold signal required to induce dauer development. Rather than repeating the entire scan, perhaps the authors could take their most discordant RILs and repeat this assay on them? It may produce a stronger signal, or at least confirm the initial screen.

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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18 Jan 2016 | for Version 1

Erik C. Andersen, Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA

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Approved With Reservations

Using techniques published previously, the authors measure the number of dauer larvae present at food patch exhaustion for a collection of Caenorhabditis briggsae recombinant lines. The dauer phenotype results are very noisy (as observed previously), but after data transformation quantitative trait loci (QTL) are detected using composite interval mapping. These results suggest that some regions of the C. briggsae genome confer differences in dauer formation, but more analysis needs to be performed to know more definitively. I would soften the abstract language (e.g. "strong support") to reflect the uncertainty in the results.

Given the variability of the phenotype measurements and the small number of recombinant lines, I am surprised about the QTL results. I would have expected no QTL to be detected, as observed in the untransformed data. It would be nice to see some more analyses, including the following:

What is the broad-sense heritability of this trait for these strains? It would be good to know how much of the trait variance you might expect to map to genomic regions in this study.
What are the results of a non-parametric linkage mapping? Composite interval mapping has many caveats. Are these QTL robust to mapping technique?
The significance threshold seems low given the allele frequency skews present in this mapping population. Please explain more about how that threshold was determined and how the skews were controlled.
Can you plot the RIL phenotypes before and after transformation so we can assess how well the transformation affected the high variability of the assay?

If the detected QTL represent expected amounts of the genetic variance, are robust to mapping technique, and are significant with respect to the known allele frequency skews in this mapping population, then I believe this article offers preliminary evidence that some genomic regions confer differences in dauer larvae formation. These results suggest genomic regions to test for causality in the trait of interest, including regions that could lead to conserved interspecific variation between C. elegans and C. briggsae.

Competing Interests

No competing interests were disclosed.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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[1] 1. Félix MA, Braendle C: The natural history of Caenorhabditis elegans. Curr Biol. 2010; 20(22): R965–R969. PubMed Abstract | Publisher Full Text

[2] 2. Petersen C, Dirksen P, Schulenburg H: Why we need more ecology for genetic models such as C. elegans. Trends Genet. 2015; 31(3): 120–127. PubMed Abstract | Publisher Full Text

[3] 3. Frézal L, Félix MA: C. elegans outside the Petri dish. eLife. 2015; 4: e05849. PubMed Abstract | Publisher Full Text | Free Full Text

[4] 4. Kiontke KC, Félix MA, Ailion M, et al.: A phylogeny and molecular barcodes for Caenorhabditis, with numerous new species from rotting fruits. BMC Evol Biol. 2011; 11(1): 339. PubMed Abstract | Publisher Full Text | Free Full Text

[5] 5. Green JW, Harvey SC: Development of Caenorhabditis elegans dauer larvae in growing populations. Nematology. 2012; 14(2): 165–173. Publisher Full Text

[6] 6. Green JW, Snoek LB, Kammenga JE, et al.: Genetic mapping of variation in dauer larvae development in growing populations of Caenorhabditis elegans. Heredity (Edinb). 2013; 111(4): 306–313. PubMed Abstract | Publisher Full Text | Free Full Text

[7] 7. Green JW, Stastna JJ, Orbidans HE, et al.: Highly polygenic variation in environmental perception determines dauer larvae formation in growing populations of Caenorhabditis elegans. PLoS One. 2014; 9(11): e112830. PubMed Abstract | Publisher Full Text | Free Full Text

[8] 8. Félix MA, Duveau F: Population dynamics and habitat sharing of natural populations of Caenorhabditis elegans and C. briggsae. BMC Biol. 2012; 10(1): 59. PubMed Abstract | Publisher Full Text | Free Full Text

[9] 9. Ross JA, Koboldt DC, Staisch JE, et al.: Caenorhabditis briggsae recombinant inbred line genotypes reveal inter-strain incompatibility and the evolution of recombination. PLoS Genet. 2011; 7(7): e1002174. PubMed Abstract | Publisher Full Text | Free Full Text

[10] 10. Stiernagle T: Maintenance of C. elegans. (february 11, 2006), WormBook. ed. the C. elegans Research Community, WormBook, 2006. PubMed Abstract | Publisher Full Text

[11] 11. Wang SC, Basten CJ, Zeng ZB: Windows QTL Cartographer 2.5. Department of Statistics, North Carolina State University, Raleigh, NC, 2007. Reference Source

[12] 12. Green J, Harvey S: Dataset 1 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015a. Data Source

[13] 13. Green J, Harvey S: Dataset 2 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015b. Data Source

[14] 14. Green J, Harvey S: Dataset 3 in: Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae. F1000Research. 2015c. Data Source

Quantitative trait loci mapping of dauer larvae development in growing populations of Caenorhabditis briggsae

Abstract

Keywords

Introduction

Methods

Results

Table 1. QTLs affecting the number of dauer larvae at food exhaustion.

Figure 1. Dauer larvae formation is variable across experimental blocks.

Figure 2. Quantitative trait mapping identifies regions affecting the number of dauer larvae at food exhaustion.

Discussion

Data availability

Author contributions

Competing interests

Grant information

Acknowledgements

References

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