Genotyping (see Supplementary information 7.2.2) and Bulked segregant analysis (see Supplementary information 7.2.3)
The next step is the genotyping of the mapping population. For this we need the DNA sequence to detect genetic variation among the plants of the mapping population.
- Q: Which plants of the population were sequenced?
- Q: Which method was used for sequencing?
The sequences obtained from the bitter and sweet parent and the sweet F2 offspring were aligned and compared to the reference genome. Variation in the DNA was monitored and the most reliable DNA variation was selected. Positions in the DNA for which the two parents were homozygous and polymorphic (e.g. parent 1: 0/0 & parent 2: 1/1) were used for genotyping the mapping population. The plants in the mapping population can be homozygous (0/0 or 1/1) or heterozygous (1/0). These variant positions are the SNP markers used for genotyping.
- Q: what are SNPs?
For genotyping a bulk of DNA sequences from the 94 sweet plants was made and for each marker the frequency of respectively Atlas and Carina red alleles was counted. So, the sequences of all 94 combined F2 plants were analysed as one bulk sample. Next, for each SNP marker the number of sequences with respectively sweet and bitter alleles were counted and a frequency of sweet and bitter alleles was obtained in the bulk of sweet progeny.
Study Figure 1:
Each dot in the figure is representing a marker locus. Alternating red and blue dots correspond to alternating chromosomes. On the Y axis is the % difference in allele frequency sweet versus bitter allele in the bulk of sweet plants, for each marker locus. So, if for instance 40% of the sequence reads has the sweet allele, 60% has the bitter allele. In the bulk of sweet plants there are several markers that have an increased frequency of alleles from the sweet parent.
- Q: On which chromosome are these markers located?
Figure 1 (click to enlarge)
The markers correlated with sweetness (100% sweet alleles in the sweet bulks) are located in the Quinoa genome on Scaffold 3489 between position 1934 and 688,027 (686 kbp) (chrom 16, see Fig 2). More precisely, a variant allele closely linked to sweet was detected in reads from position 350,000-360,000
- Q: Follow the link to the Chenopodium quinoa v1.0 genome on http://www.phytozome.net/
- Q: Find the genome (in unclustered genomes).
- Q: Find Scaffold 3489 in the genome with Jbrowse
- Q: What is the size of the scaffold?
- Q: Go to Scaffold 3489, position 350,000-360,000 and see which transcript is located in this region.
- Q: What is the name of this mRNA?
- Q: Find out more details of this gene (right mouse click, while standing on the transcript)
Remark: If you like you can try the same in another browser at
http://www.cbrc.kaust.edu.sa/chenopodiumdb/ and compare both genome browsers
Figure 2 (click to enlarge)
bHLH genes are known to regulate triterpenoid biosynthesis in Medicago truncatula. These HLH transcription factors bind to a DNA motif 5’-CACGHG-3’. This DNA motif is found in the promoter of several of Quinoa genes acting in the saponin biosynthesis route. So, this gene can regulate the expression of other genes, for instance genes in the saponin biosynthesis route (see figure 4: saponin biosynthesis route). Furthermore, in the sweet lines specific DNA variation is found within this gene. This variation is introducing a cryptic splice site that generates a premature stop-codon, and thereby alters the protein in sweet lines (see Figure 3).
- Q: What are the arguments for AUR62017204 as a candidate gene?
Figure 3 (click to enlarge)
Figure 4: Saponin biosynthesis pathway (click to enlarge)
Differential expression/BSA (7.1.5)
RNA was isolated from inflorescences of individual plants (parents and 45 x F3 progeny plants) containing a mixture of flowers and seeds at various stages of development. Furthermore, it has to be noted that the presence and absence of saponins was correlated with differences in seed coat thickness, with the bitter lines having significantly thicker seed coats than sweet lines. Genes differentially expressed between bitter and sweet lines (population Kurmi (sweet) x 0654) were identified and the fold change in expression between sweet and bitter progeny is listed (See File: Expression differences SWEET x BITTER.xlsx). Please download the file, don't use the preview.
- Q: Find the candidate gene AUR62017204 (C_Quinoa_Scaffold_3489:353903-356143) in this list of differentially expressed genes. Is the gene up- or down-regulated in the sweet progeny?
- Q: The sweet lines carry a non-functional allele of AUR62017204. Study the saponin biosynthesis pathway in figure 2. Study the differential expression .xlsx file and find five genes that are acting in the saponin biosynthesis pathway that are differentially expressed.
- Q: Are they up or down regulated in the sweet lines?
- Q: Explain how the DNA polymorphism in the candidate gene at the QTL locus can influence the expression saponin biosynthesis in the sweet lines.
- Q: Can you think of a reason why, besides the saponin pathway genes, also other genes are differentially expressed?
http://www.cbrc.kaust.edu.sa/chenopodiumdb/
Genotyping (see Supplementary information 7.2.2) and Bulked segregant analysis (see Supplementary information 7.2.3)
The next step is the genotyping of the mapping population. For this we need the DNA sequence to detect genetic variation among the plants of the mapping population.
- Q: Which plants of the population were sequenced?
- Q: Which method was used for sequencing?
The sequences obtained from the bitter and sweet parent and the sweet F2 offspring were aligned and compared to the reference genome. Variation in the DNA was monitored and the most reliable DNA variation was selected. Positions in the DNA for which the two parents were homozygous and polymorphic (e.g. parent 1: 0/0 & parent 2: 1/1) were used for genotyping the mapping population. The plants in the mapping population can be homozygous (0/0 or 1/1) or heterozygous (1/0). These variant positions are the SNP markers used for genotyping.
- Q: what are SNPs?
For genotyping a bulk of DNA sequences from the 94 sweet plants was made and for each marker the frequency of respectively Atlas and Carina red alleles was counted. So, the sequences of all 94 combined F2 plants were analysed as one bulk sample. Next, for each SNP marker the number of sequences with respectively sweet and bitter alleles were counted and a frequency of sweet and bitter alleles was obtained in the bulk of sweet progeny.
Study Figure 1:
Each dot in the figure is representing a marker locus. Alternating red and blue dots correspond to alternating chromosomes. On the Y axis is the % difference in allele frequency sweet versus bitter allele in the bulk of sweet plants, for each marker locus. So, if for instance 40% of the sequence reads has the sweet allele, 60% has the bitter allele. In the bulk of sweet plants there are several markers that have an increased frequency of alleles from the sweet parent.
- Q: On which chromosome are these markers located?
Figure 1 (click to enlarge)
The markers correlated with sweetness (100% sweet alleles in the sweet bulks) are located in the Quinoa genome on Scaffold 3489 between position 1934 and 688,027 (686 kbp) (chrom 16, see Fig 2). More precisely, a variant allele closely linked to sweet was detected in reads from position 350,000-360,000
- Q: Follow the link to the Chenopodium quinoa v1.0 genome on http://www.phytozome.net/
- Q: Find the genome (in unclustered genomes).
- Q: Find Scaffold 3489 in the genome with Jbrowse
- Q: What is the size of the scaffold?
- Q: Go to Scaffold 3489, position 350,000-360,000 and see which transcript is located in this region.
- Q: What is the name of this mRNA?
- Q: Find out more details of this gene (right mouse click, while standing on the transcript)
Remark: If you like you can try the same in another browser at
http://www.cbrc.kaust.edu.sa/chenopodiumdb/ and compare both genome browsers
Figure 2 (click to enlarge)
bHLH genes are known to regulate triterpenoid biosynthesis in Medicago truncatula. These HLH transcription factors bind to a DNA motif 5’-CACGHG-3’. This DNA motif is found in the promoter of several of Quinoa genes acting in the saponin biosynthesis route. So, this gene can regulate the expression of other genes, for instance genes in the saponin biosynthesis route (see figure 4: saponin biosynthesis route). Furthermore, in the sweet lines specific DNA variation is found within this gene. This variation is introducing a cryptic splice site that generates a premature stop-codon, and thereby alters the protein in sweet lines (see Figure 3).
- Q: What are the arguments for AUR62017204 as a candidate gene?
Figure 3 (click to enlarge)
Figure 4: Saponin biosynthesis pathway (click to enlarge)
Differential expression/BSA (7.1.5)
RNA was isolated from inflorescences of individual plants (parents and 45 x F3 progeny plants) containing a mixture of flowers and seeds at various stages of development. Furthermore, it has to be noted that the presence and absence of saponins was correlated with differences in seed coat thickness, with the bitter lines having significantly thicker seed coats than sweet lines. Genes differentially expressed between bitter and sweet lines (population Kurmi (sweet) x 0654) were identified and the fold change in expression between sweet and bitter progeny is listed (See File: Expression differences SWEET x BITTER.xlsx). Please download the file, don't use the preview.
- Q: Find the candidate gene AUR62017204 (C_Quinoa_Scaffold_3489:353903-356143) in this list of differentially expressed genes. Is the gene up- or down-regulated in the sweet progeny?
- Q: The sweet lines carry a non-functional allele of AUR62017204. Study the saponin biosynthesis pathway in figure 2. Study the differential expression .xlsx file and find five genes that are acting in the saponin biosynthesis pathway that are differentially expressed.
- Q: Are they up or down regulated in the sweet lines?
- Q: Explain how the DNA polymorphism in the candidate gene at the QTL locus can influence the expression saponin biosynthesis in the sweet lines.
- Q: Can you think of a reason why, besides the saponin pathway genes, also other genes are differentially expressed?
http://www.cbrc.kaust.edu.sa/chenopodiumdb/