ANS/PLSS 433: Molecular Breeding 1

I.	RFLP MAPPING AND BREEDING APPLICATIONS

Introduction:

     Improved Genetics resulting from cross breeding accounts for 50% of 
the yearly increase in agricultural productivity. Improvements are achieved 
by picking good (pheno)types. In plants we pick high yield, disease 
resistance, adaptability and quality. In animals we pick reproductive 
efficiency, feed efficiency, disease resistance and product yield and 
quality. Now for the first time in 50,000 yr of human farming and husbandry 
we can pick genes (genotypes) not just phenotypes using a gene probe and 
Southern blot or other molecular marker.

     We saw that using a Southern blot and a radioactive beta globin gene 
probe we could detect an RFLP linked to sickle cell anaemia. This is a rare 
example where we already cloned an important gene. Very few important 
genes have been cloned. The genes we have cloned tend to encode proteins 
that are 

	i. 	highly abundant or
	ii. 	show assayable enzyme activity in vitro or
	iii. 	will complement bacterial or yeast mutants or
	iv.	confer a herbicide or antibiotic resistance on 
		transformants.
    
	Mostly we infer the existence of important genes by a phenotype 
which is genetically inherited in a Mendelian fashion and then define its 
position on a chromosome by linkage to several RFLP markers from a randomly 
selected set of 100-200 tested! HOW? Lets review genetic principles. 

Mendelian Genetics:

(see the Cartoon Guide pg 37 - 78)

1. Dominance - Genes control plant size. When a tall parent (AA) is crossed 
	with a short parent (aa) the offspring (Aa) are all tall!
	When the offspring (Aa) are selfed or crossed their offspring are 
	(AA) (Aa) (Aa) (aa) or 3 tall 1 short.

2. Independent Assortment - Genes on separate chromosomes assort 
	independently eg height and smoothness.

3. Linkage - Genes which do not assort independently are linked on 
	chromosomes eg. hairy or dwarf tomatoes.

4. Crossing over - causes linked genes segregate abnormally with a 
	frequency in a progeny population proportional to their distance 
	apart on a chromosome.

5. RFLP markers assort exactly like genes, not surprisingly since they 
	represent the DNA sequence, the stuff that genes are made of.

Gene Mapping:

	Gene mapping is simply a set of cross over frequencies between 
genes. Gene mapping is limited by the number of phenotypes we can 
recognize as different between two parents BUT there are millions of 
differences in their gene sequence which can be detected as RFLPs by 
randomly isolated gene probes. RFLPs are generated by insertions or 
deletions or sequence changes, these are very frequent events in 
non-coding DNA. 

Summary:

	RFLP probes closely linked to important genes can serve as markers 
for those important genes and thereby aid breeding programs and even allow 
a new route to genetic gene cloning.

II.	MORE RFLP MAPPING AND BREEDING APPLICATIONS

Introduction:
 
     Most useful agriculturally genes have not yet been cloned. We reviewed 
Mendelian genetics because the predictions of Mendels laws allows us to 
identify useful genes and map them by linkage to one another. This 
predictable behavior of genes underpins the science of breeding. There are 
a limited number of genes we can recognize by phenotype. However molecular 
probes are unlimited in number and as the sickle cell anaemia example shows 
we can identify parental types and hybrid types in any population. Put 
these two ideas together and you have RFLP mapping to aid  breeding.

Libraries of Cloned Probes:

	Small pieces of cloned chromosomal DNA are used individually to 
detect a characteristic restriction fragment by Southern Hybridization. 
To map a whole genome a set of about 500 random clones are prepared for 
use as probes, this set is called a library. DNA isolated from the species 
of interest is digested with restriction enzyme to 2-5 kbp. To isolate 
individual fragments from the mix we clone this DNA. Individual DNA 
fragment molecules are ligated into separate bacterial plasmid molecules. 
Each pair of ligated molecules transforms a bacterium to antibiotic 
resistance carried on the plasmid molecule. Each transformed cell grows 
into a single colony, which we can grow further and store as a new strain! 
Plasmid DNA extracted from this strain contains 1 restriction fragment 
form the 10,000,000 in the original ligation. This individual restriction 
fragment or gene can be labelled with radioactivity and used as a probe in 
a Southern blot.

Construction of RFLP Genetic Maps:

	Recombination between homologous chromosomes is the basis of 
genetic mapping. Recombination frequency between genes is a function of 
their distance apart. So map distance is a function of recombination 
during the meiosis of gamete formation.

RFLP inheritance Patterns:

	Compare an RFLP markers with Flower color.  The first marker is 
closely linked.  The second marker is linked but shows 25 % recombination 
and so is 25 cM away.  The third marker is unlinked, it shows 
50% recombination.  If we compile information with 100 or more probe we 
would have a map of the entire genome covering 1000-2000 cM with probes 
less than 10 cM apart. (1 cM = 600 Kbp approximately).