An example of F1 Verification | Application of Molecular Marker Technology to QA/QC in Plant Breeding

MNC358/PAN127
3 markers selected based on the previous polymorphism check
F1-4 result of selfing, not a cross

SNP	MNC358	PAN127	F1-1	F1-2	F1-3	F1-4	F1-5
BARC-PV-0001385	C:C	T:T	C:T	C:T	C:T	C:C	C:T
BARC-PV-0002100	G:G	A:A	A:G	A:G	A:G	G:G	A:G
BARC-PV-0002984	A:A	G:G	A:G	A:G	A:G	A:A	A:G

Table 5: The table above shows the genotyping results of three SNP markers tested on five F1 lines derived from a cross between the two parental lines MNC358 and PAN127. Recall that F1s should always be heterozygotes, containing an allele inherited from each parent. The line F1-4 is homozygous at all three SNP markers, and therefore is clearly the result of a self-pollination, not a cross between these two parents [data provided by Hamer Paschal]

Other verification examples

Backcrossing

Backcrossing is another application where molecular markers can affirm that the plants are the result of the correct cross. There are two common strategies for using markers to confirm QA/QC:

Verify initial F1s
Conduct background and foreground selection in BC1 to BCn

Verification of Identity of Breeding Populations

The identity of any breeding population can be confirmed using markers as well. As there may be too many populations to verify all, one can either conduct random spot checks using 20-30 SNP markers, check only priority lines, or check only when an error is suspected.

The marker results should be compared to the previous parental fingerprint data to help identify anomalies.

Verification of Lines in Yield Trials

In yield trials, it is a good idea to verify lines at this final stage as well. Here are a few suggested guidelines to do that:

Use 20 to 30 SNP markers
Conduct spot checks on lines using samples taken from the same line in different replications and locations
Prioritize
- Lines in 2nd year yield trials and more advanced depending on size of program
Routine fingerprinting
- 2nd year yield trial entries every year

Example of Line Verification

Let's now follow a case study to apply these concepts. The table below shows the genotyping results of two lines derived from a cross between parents CAL143 and G2333. Three samples of each of the two lines from three different locations were tested with 20 SNP markers and compared to the parental genotypes. Sixteen of the SNPs were polymorphic between the parents. We can see that there is a problem with Line 2 at the second location, as its genotype does not match the parents nor the same lines grown in the other locations [data provided by Hamer Paschal].

Table 6: Data from markers used to confirm lines derived from controlled crosses.

2 lines derived from CAL143/G2333 planted at 3 locations
20 SNP markers
16 polymorphic markers between parents
Coefficient of Similarity (CoS) of parents = 20%
Here there is clearly an error in Line 2 planted at Location 2 as it does not resemble the other samples of this line

				Line 1			Line 2
SNP	CAL143	G2333	Loc 1	Loc 2	Loc 3	Loc 1	Loc 2	Loc 3
BARC-PV-0000055	A:A	T:T	A:A	A:A	A:A	T:T	G:G	T:T
BARC-PV-0000105	C:C	A:A	A:A	A:A	A:A	C:C	T:T	C:C
BARC-PV-0000365	G:G	T:T	T:T	T:T	T:T	T:T	A:A	T:T
BARC-PV-0000468	A:A	T:T	T:T	T:T	T:T	A:A	C:C	A:A
BARC-PV-0000501	T:T	G:G	G:G	G:G	G:G	G:G	G:G	G:G
BARC-PV-0000623	G:G	A:A	A:A	A:A	A:A	A:A	T:T	A:A
BARC-PV-0000669	A:A	T:T	T:T	T:T	T:T	T:T	A:A	T:T
BARC-PV-0000819	A:A	G:G	G:G	G:G	G:G	A:A	C:C	A:A
BARC-PV-0000909	G:G	C:C	C:C	C:C	C:C	C:C	G:G	C:C
BARC-PV-0001361	C:C	T:T	T:T	T:T	T:T	T:T	C:C	T:T
BARC-PV-0001385	C:C	C:C	C:C	C:C	C:C	C:C	C:C	C:C
BARC-PV-0001413	T:T	T:T	T:T	T:T	T:T	T:T	A:A	T:T
BARC-PV-0001591	T:T	C:C	T:T	T:T	T:T	C:C	G:G	C:C
BARC-PV-0001925	T:T	G:G	G:G	G:G	G:G	G:G	T:T	G:G
BARC-PV-0002061	G:G	A:A	A:A	A:A	A:A	A:A	C:C	A:A
BARC-PV-0002064	G:G	G:G	G:G	G:G	G:G	G:G	A:A	G:G
BARC-PV-0002100	G:G	A:A	A:A	A:A	A:A	A:A	C:C	A:A
BARC-PV-0002435	T:T	T:T	G:G	G:G	G:G	T:T	G:G	T:T
BARC-PV-0002440	G:G	C:C	C:C	C:C	C:C	C:C	C:C	C:C
BARC-PV-0002776	C:C	T:T	T:T	T:T	T:T	C:C	A:A	C:C