Detecting ERECTA Gene Using PCR and QTL Analysis

Detection of ERECTA gene and QTL analysis of RI lines of Arabidopsis thaliana (Landsberg erecta (Ler-0) and Columbia (Col-0))

Practical Exercises in Plant Breeding

• Mohammad Foteh Ali

 

1. Abstract: Recombinant inbreed (RI) lines of Arabidopsis thaliana have been crossed between the ecotypes Landsberg erecta (Ler-0) and Columbia (Col-0) up to 8th generation. The main aim of this laboratory report is the detection of ERECTA gene using PCR and QTL analysis for different traits. Phenotypic analysis of 42 RI lines and genotypic analysis using markers suggest that m220 is very closed to ERECTA gene. QTL analysis also showed that different position of chromosome 2 are responsible for different phenotypic traits.

2. Introduction

Fig 1: Arabidopsis thaliana Plant

Arabidopsis thaliana is a well-accepted experimental plant in plant biology & also popular model organism in genetics. Arabidopsis is a small flowering plant. It posses short life cycle with 2n=10 genotypes. Arabidopsis is under Brassicaceae family .Even though, most of the members of this family have agronomic importance but in plant molecular biology this plant is mainly used to conduct basic research and also plays foremost role in quantitative genetics (Bevan M, Walsh S. 2005).

This lab work was conducted to identify the genotype of Recombinant Inbreeding Line (RIL) using known PCR markers and also QTL analysis of those markers was also done. Landsberg erecta and Colombia were used as Arabidopsis plant materials to develop Recombinant Inbreeding Lines (RIL). Ecotypes of Landsberg erecta and Colombia were segregated up to eight times to increase more homozygote and to develop Recombinant Inbreeding Lines (RIL) (Lister and Dean 1993). In this exercise, 42 RILs were used with high recombination frequency. For each RIL as well as parental line, some phenotypic traits were evaluated to identify recombination frequency. The main aim of this exercise is to identify the position of ERECTA gene by mapping the RILs. Genotypes of molecular markers of these RI lines were well categorized, and they have been used in several QTL analysis. From phenotypic trait table we calculated score of markers, and the markers having the high correlation gave the high position of ERECTA gene. Computational analysis was also performed for mapping quantitative trait loci (QTL) of different chromosomes.

3. Materials and Methods

3. 1 Plant materials:

Segregated Recombinant Inbred lines (RI lines) at the F8 generation of Arabidopsis thaliana were obtained by crossing between Landsberg erecta (Ler-0) and Columbia (Col-0). The seeds were kept on 1 MS medium with 0.8%agar for germination. Germinated small plants were transferred into commercial potting soil medium for growth and development. Parental line of Landsberg erecta (Ler-0) and Columbia (Col-0) also planted in soil. But RIL CS/1960 was not transplanted due to lack of germination. The plants were kept in green house at 18º night and 22 º day temperatures.

3.2 DNA extraction:

For DNA extraction 2 leaves of each RI lines of CS/N1900, CS/N1903, CS/N1911, CS/N1915, CS/N1921, CS/N1927, CS/N1951 and Landsberg erecta (Ler-0) and Columbia (Col-0) were also used as parent materials . The extraction was performed with a column based Qiagen DNeasy Plant Mini Kit protocol. For details DNA extraction procedure we followed our laboratory manual with two exception. We were used two leaves instead of one and we used tissue lyser instead of grinding.

3.3 PCR setup

For PCR results we had to conduct 10 reactions. The PCR setup was followed by laboratory manual. Marker m555 was taken to complete our group work. The cycling conditions of the PCR was as follows: 94 °C for 5 min, 35 cycles of 94 °C for 30 s, 57 °C for 30 s, 72 °C for 30 s; 72 °C for 7 min and 4 °C.

3.4 Agarose gel Electrophoresis:

1.5% agarose gel was prepared by mixing 3g agarose with 200ml TBE buffer in a 250ml bottle. The solution was heated properly until obtaining an uniform mixture of all pieces of agarose floating around. Then it was kept in a 55º cabinet. 100 µl of ethidium bromide (from 0.5mg/ml stock solution) was added to agarose gel in a 250ml bottle. DNA ladders were put into the tray& then gel was added. After removing the ladders PCR samples were added. 80 V electricity was passed through the gel for 30min then 150v for 20 min. Finally picture was taken with gel camera.

4. Result

4.1 Genotype analysis

DNA extraction of RI lines CS/N 19( 00,03,11, 15,21,27,51) showed different PCR band by gel camera image. As illustrated in Fig.2 we used PCR based marker m555 for genotype analysis. But we found only RIL 21 as Colombia(Col-0), RIL 27 as Landsberg erecta (Ler-0) and RIL 51 as Columbia (Col-0). Fig.2 showed that between Colombia(Col-0) and Landsberg erecta, there had distinct difference . The most visible band was Landsberg erecta (Ler-0) and the band which was laid on the same base pair position was Landsberg erecta (Ler-0) and others were Colombia(Col-0) .

Agarose gel electrophoresis helped analysis the band of PCR sample among different RI lines. We also collected other band from all markers (Tab.1). Table 1 shows that result of PCR followed by Agarose gel electrophoresis. Water does not show any result as expected. We also missed some result of PCR band and most of the result of PCR band is correlated with parental line.

Table 1. Results of genotypes from the agarose gel electrophoresis.

PCR			RIL CS/N 1900	RIL CS/N 1903	RIL CS/N 1911	RIL CS/N 1915	RIL CS/N 1921	RIL CS/N 1927	RIL CS/N 1951
marker	Col-0	Ler	H2O
m213	Col	Ler	Col	–	Col	Col	Col	Col	Ler	–
m251	Col	Ler	Ler	Col	Col	Col	Ler	Ler	Col	–
ERECTA	Col	Ler	Ler	Col	Col	Ler	Ler	Ler	Ler	–
m220	Col	–	Ler	Col	–	Ler	Ler	Ler	Ler	–
m457	Col	Ler	Col	Col	Ler	Col	Col	Col	Col	–
m600	Col	Ler	Col	Col	Ler	Ler	Col	Col	Ler	–
m555	–	Ler	–	–	–	–	Col	Ler	Col	–
m336	Col	Ler	Ler	Col	Col	Col	Col	Ler	Ler	–
Phenotype	Col	Ler	Ler	Col	Col	Ler	Ler	Ler	Ler	–

Col= Columbia (Co0), Ler= Landsberg erecta (Ler-0)

Based on Table.1 we calculate the correlation between markers and phenotypes. ERECTA is highest correlated as expected (Sum of agreement: 9,100%). The highly correlated markers are m220 (7,78%), m251 (7,78%), and m336 (7, 78%) and they all are situated in chromosome 2. The result of other markers are respectively as m213(4,44%), m457(3,33%), m600(5,56%) and m555 (3, 33%).

4.2 Phenotypic analysis:

Phenotypic traits of Columbia (Col-0) and Landsberg erecta (Ler-0) were analysed on the basis of height, stem thickness, leaf shape, pedicle, inflorescence, silique, of growing plants. Among 40(CS/n1960 didn’t germinated and CS/N1985 died at seedling stage) RI lines we found 27 RI lines of Landsberg erecta (Ler-0) and 13 RI lines of Columbia (Col-0) (Appendix 1).

Table 2: 25 Markers and 40 identified phenotypes correlation

	m105	m213	m216	m217	m220	m226	m246	m247	m247	m251	m253	m291	m315
Correlation	23	14	28	21	34	27	24	20	17	34	18.5	23.5	16
Correlation%	58	35	68	53	85	68	58	50	42.5	85	46.3	58	40

	m326	m336	m424	m435	m457	488	m506	m518	m532	m555	m583	m600
Correlation	27	25	20	22	15	25	16	21	18	17	21.5	27.5
Correlation%	66	63	50	55	38	63	40	50	45	43	53.8	68.8

After scoring(Tab:2), we found m251 and m220 had the highest correlation(34,85%) value. These are the highest value of markers and both are situated on chromosome 2. From this result we can assume that ERECTA gene may be situated between m251 and m220.

4.3 QTL analysis:

Table 3: QTL result for different trait detected by Interval Mapping methods.  

Trait	Chromosome	cM	Lod	Additive
Height	2	30.6	3.42	-4.33
Stem thickness	2	21.7	3.3	0.30
	2	37.0	6.06	0.35
Compact inflorescence	2	22.7	5.71	-0.37
	2	37.5	6.10	-0.37
Silique length	no QTL	no QTL	no QTL	no QTL
Silique width	2	21.7	6.07	0.46
	2	37.5	5.13	0.49
Pedicile length	2	26.4	9.91	-3.15
Leaf shape	2	13.2	3.49	0.34
	2	22.2	5.74	0.43
	2	35.5	3.77	0.34
Col_Ler	2	21.1	5.77	0.45
	2	37.5	6.14	0.39

cM=centimorgan, LOD= logarithms of odd

In QTL analysis (Tab:3), we performed interval mapping (IM) for RI lines. We found highest peak(Fig:3) for height in chromosome 2 with approximately 30.6 centimorgan (cM),logarithms of odds (LOD) 3.2 and additive effects -4.3. In case of stem thickness, we found 2 peaks at chromosome 2 with highest 37cM, 6.06 LOD and additive effects 0 .35. In case of compact inflorescence the highest peak in chromosome 2 with 37.5cM, 6.10LOD,-0.37 additive effects. We didn’t found any QTL in silique length. In silique width, highest peak was 37.5cM at chromosome 2 with 5.13LOD and 0.49 additive effects. In leaf shape, we had three peaks and in pedicle length we found highest 9.91 LOD with 22.5cM peak value .In selection of Ler/Col we found 37.5cM peaks value and 6.14 LOD with 0.39 additive effects.

Fig 3: LOD score curves of all chromosome for different phenotypic trait derived from a cross between Ler and Col. The curves derived from the Interval mapping(IM). The horizontal line point out as thresh hold level.

5. Discussion

To decrease any secondary effects caused by transferring the medium, all the RI lines grew in the same solution using culture apparatuses (Toda et al. 1999a). RI line CS/N 1960 didn’t germinate due to lack of germination and CS/N 1985 died at seedlings stage. We can grow plants in more environmental control conditions to avoid death of plants. The gel electrophoresis or PCR result gives weak and no band for different RI lines. The main cause of this problem is not the DNA template, because others groups found clear band on PCR results. It may be the pipetting error at the time of loading DNA on agrose gel. We found only Landsberg erecta as parental line . On the basis of this position we assume the another band on the same position of CS/N 1927 was Landsberg erecta (Ler-0) and others were Columbia (Col-0). From PCR result and phenotypic analysis we found markers m220 (7,78%), m251 (7,78%), and m336 (7, 78%) had highest correlation and all are situated in chromosome 2. Markers m213, m457, m600 they have lowest correlation value and they situated on different chromosomes. We found few band in m555 (3) due to pipetting error but we can skip this result because this markers placed on chromosome no 5. For more accurate result we calculate the correlation of phenotypes of 40 RI lines and 25 markers. At the time of phenotype analysis we found some variation on leave shape for environmental effect (Alonso-Blanco, C. 2000). The result showed that m220 and m251 had highest value. We can accept this result because we used more population in this analysis. So we can suppose that ERECTA gene is located between m220 and m 251. To get more accurate result one repeat the PCR analysis.

For QTL analysis we performed Interval Mapping (IM) method because this method is more accurate than the others. RI lines provides a good materials for QTL (Knapp and Bridges 1990). Different traits like height, stem, inflorescence, silique, pedicle, and leaf used for QTL. In height and pedicle length we found one peak above thresh hold level but others had two peaks and in silique length we did not found any QTL. In case of two peaks we can suggest the highest cM is the position of QTL of these trait. But for more accurate QTL one can do more accurate phenotypic analysis. So the results from QTL indicates the position of responsible gene for specific traits.

Appendix 1

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Line	Height (cm)	Stem thickness	Compact inflorescence	Silique length (mm)	Silique width	Pedicle length (mm)	Leaf shape	Petiole length (mm)	Col/Ler
CS/N1900	19	thick	compact	12	thick	6	no serration		Ler
CS/N1901	22	thin	not compact	10	thin	8	serration		Col
CS/N1903	26	thin	not compact	15	thin	10	serration		Col
CS/N1905	22	thin	not compact	17	thin	11	serration		Col
CS/N1910	12.5	thick	compact	10	thick	4	no serration		Ler
CS/N1911	23	thin	not compact	10	thin	7	serration		Col
CS/N1913	7	thick	compact	10	thick	5	no serration		Ler
CS/N1915	17.5	thick	compact	15	thick	3	no serration		Ler
CS/N1919	15	thick	compact	10	thick	3	no serration		Ler
CS/N1921	10	thick	compact	13	thick	4	no serration		Ler
CS/N1924	25	thin	not compact	16	thin	12	serration		Col
CS/N1927	12.5	thick	compact	7	thick	5	no serration		Ler
CS/N1929	25	thin	not compact	10	thin	12	serration		Col
CS/N1933	9.5	thick	compact	13	thick	4	no serration		Ler
CS/N1934	14.5	thick	compact	11	thick	5	no serration		Ler
CS/N1935	14.5	thick	compact	11	thick	4	no serration		Ler
CS/N1937	19	thick	compact	9	thick	3	no serration		Ler
CS/N1938	20	thick	compact	12	thick	4	no serration		Ler
CS/N1942	17	thick	compact	10	thick	5	no serration		Ler
CS/N1945	24	thin	not compact	10	thin	13	serration		Col
CS/N1946	22	thick	not compact	15	thin	12	serration		Col
CS/N1948	28	thin	not compact	15	thin	10	no serration		Col
CS/N1951	3	thick	compact				no serration		Ler	no siliques
CS/N1953	26	thin	not compact	10	thin	11	no serration		Col
CS/N1954	30	thin	not compact	8	thin	6	serration		Col
CS/N1957	17.5	thick	compact	12	thick	4	no serration		Ler
CS/N1958	15.5	thick	compact	11	thick	5	no serration		Ler
CS/N1959	14	thick	compact	15	thick	4	no serration		Ler
CS/N1960										no plant
CS/N1963	17.5	thick	compact	10	thick	4	no serration		Ler
CS/N1966	15	thick	compact	13	thick	4	no serration		Ler
CS/N1969	11	thick	compact	11	thick	2	no serration		Ler
CS/N1970	16.5	thick	compact	13	thick	4	no serration		Ler
CS/N1971	25	thin	not compact	15	thin	6	serration		Col
CS/N1974	19