Expanded tomato chromosome map and cDNA library

Claim 2

A cDNA library corresponding to tomato DNA comprising at least two clones selected from the group consisting of CD10, CD76, CD23, CD53, CD66, CD71, CD69, CD68, CD36, CD70, CD78, CD74, CD26, CD67, CD72, CD18, CD17, CD19, DC39 and CD2.

Claim 3

The cDNA library of claim 1 wherein said two clones are selected from the same chromosome.

Claim 4

The cDNA library of claim 1 containing all the clones listed in claim 1.

Claim 5

A cDNA clone selected from the group consisting of CD10, CD76, CD23, CD53, CD66, CD71, CD69, CD68, CD36, CD70, CD78, CD74, CD26, CD67, CD72, CD18, CD17, CD19, CD39 and CD2.

Claim 6

A cDNA clone selected from the group consisting of CD10, CD76, CD23, CD53, CD66, CD71, CD69, CD68, CD36, CD70, CD78, CD74, CD26, CD67, CD72, CD18, CD17, CD19, CD39 and CD2.

Claim 7

A plasmid containing a cDNA clone of claim 4.

Claim 8

The plasmid of claim 5 comprising pBR322 or pUC8.

Claim 9

A chromosome map useful for determining the original position or positions of a tomato DNA fragment on the tomato chromosome comprising a representation of at least one chromosome with markers spaced therealong in sequential order with spacing given in map units (cM) as follows: chromosome 1: Idh-1 - 18cM - Prx-1 - 21cM - CD15 - 7cM - CD10 and/or CD76 - 11cM - Skd-1 - 4cM - CD24 - 17cM - CD47 - 4cM - CD12 - 27cM - CD9B and/or CD52A - 5cM - CD9A and/or CD16B and/or CD23 - 5cM - CD16A and/or CD20 and/or act-3 - 10 cM - CD44 - 1cM - CD28 - 18cM - Est-3; chromosome 2: R45s and/or CD49B - 1cM - CD37 - 7cM - CD1 - 7cM - Rbcs-1 and/or 3-91 - 15cM - CD33A - 9cM - CD30B - 22cM - Cab-1 and/or 3-131 - 1cM - CD11 and/or CD53 - 11cM - Est-7 - 8cM - Prx-2 - 6cM - CD43 - 3cM - CD35 - 6cM - CD9C - 13cM - Rbcs-3 - 10cM - CD30A and/or CD33B - 9cM - CD66; chromosome 3: CD4A - 13cM - CD71 - 5cM - CD69 - 30cM - Cab-3 - 12cM - Pgm-1 - 18cM - CD50 - 2cM - Rbcs-2 and/or CD68 and/or 3-­167 - 6cM - CD52B and/or CD36A and/or CD31B and/or CD51 - 6cM - CD29C and/or Act-4 - 2cM - CD13A and/or CD6B; chromosome 4: 6Pgdh-1 - 11cM - CD59 - 5cM - CD49A - 11cM - Act-1 and/or Tpi-2 - 15cM - Pgm-2 and/or CD70 - 9cM - Adh-1 - 3cM - Act-10 and/or CD55 - 33cM - CD39; chromosome 5: CD41 - 9cM - CD64 - 8cM - CD31A - 10cM - CD38B and/or CD36B - 42cM - CD78 - 6cM - CD74A; chromosome 6: CD14 - 8cM - CD13B and/or CD67 - 7cM - Aps-1 - 11cM - CD29B - 13cM - Adh-2 - 5cM - CD25A - 8cM - CD42; chromosome 7: CD61 - 3cM - CD58 and/or CD65 - 8cM - CD48 - 16cM - Got-3 and/or CD26 - 7cM - Aco-2 - 2cM - Got-2 - 7cM - Cab-4 - 5cM - CD54 and/or CD57; chromosome 8: CD46 - 10cM - CD60 - 10cM - CD7 - 10cM - Cab-2 and/or 3-185 - 12cM - Aps-2 - 14cM - CD40 - 1cM - CD21A and/or CD16C; chromosome 9: Act-2 - 6cM - CD5 - 5cM - CD32B; chromosome 10: CD56 - 7cM - CD45 - 10cM - Est-8 - 5cM - CD38A - 10cM - CD34B - 10cM - CD34A and/or Act-6 - 4cM - CD72; CD32A - 11cM - cD3 - 3cM - CD8; chromosome 11: Sod1 and/or Act5 - 4cM - CD29D - 4cM - CD18 - 10cM - CD25B - 16cM - CD17; chromosome 12: CD21B - 1cM - 6Pgdh-2 and/or CD27 - 10cM - CD4B and/or CD16D and/or CD22 - 2cM - CD6A - 3cM - Pgi-1 - 38cM - CD19 - 2cM - Aco-1 - 2cM - Cab-5 and/or 3-48; CD2

Claim 10

A chromosome map useful for determining the original position or positions of a tomato DNA fragment on the tomato chromosome comprising a representation of at least one chromosome with markers spaced therealong in sequential order with spacing given in map units (cM) as follows: chromosome 1: Idh-1 - 18cM - Prx-1 - 21cM - CD15 - 7cM - CD10 and/or CD76 - 11cM - Skd-1 - 4cM - CD24 - 17cM - CD47 - 4cM - CD12 - 27cM - CD9B and/or CD52A - 5cM - CD9A and/or CD16B and/or CD23 - 5cM - CD16A and/or CD20 and/or act-3 - 10 cM - CD44 - 1cM - CD28 - 18cM - Est-3; chromosome 2: R45s and/or CD49B - 1cM - CD37 - 7cM - CD1 - 7cM - Rbcs-1 and/or 3-91 - 15cM - CD33A - 9cM - CD30B - 22cM - Cab-1 and/or 3-131 - 1cM - CD11 and/or CD53 - 11cM - Est-7 - 8cM - Prx-2 - 6cM - CD43 - 3cM - CD35 - 6cM - CD9C - 13cM - Rbcs-3 - 10cM - CD30A and/or CD33B - 9cM - CD66; chromosome 3: CD4A - 13cM - CD71 - 5cM - CD69 - 30cM - Cab-3 - 12cM - Pgm-1 - 18cM - CD50 - 2cM - Rbcs-2 and/or CD68 and/or 3-­167 - 6cM - CD52B and/or CD36A and/or CD31B and/or CD51 - 6cM - CD29C and/or Act-4 - 2cM - CD13A and/or CD6B; chromosome 4: 6Pgdh-1 - 11cM - CD59 - 5cM - CD49A - 11cM - Act-1 and/or Tpi-2 - 15cM - Pgm-2 and/or CD70 - 9cM - Adh-1 - 3cM - Act-10 and/or CD55 - 33cM - CD39; chromosome 5: CD41 - 9cM - CD64 - 8cM - CD31A - 10cM - CD38B and/or CD36B - 42cM - CD78 - 6cM - CD74A; chromosome 6: CD14 - 8cM - CD13B and/or CD67 - 7cM - Aps-1 - 11cM - CD29B - 13cM - Adh-2 - 5cM - CD25A - 8cM - CD42; chromosome 7: CD61 - 3cM - CD58 and/or CD65 - 8cM - CD48 - 16cM - Got-3 and/or CD26 - 7cM - Aco-2 - 2cM - Got-2 - 7cM - Cab-4 - 5cM - CD54 and/or CD57; chromosome 8: CD46 - 10cM - CD60 - 10cM - CD7 - 10cM - Cab-2 and/or 3-185 - 12cM - Aps-2 - 14cM - CD40 - 1cM - CD21A and/or CD16C; chromosome 9: Act-2 - 6cM - CD5 - 5cM - CD32B; chromosome 10: CD56 - 7cM - CD45 - 10cM - Est-8 - 5cM - CD38A - 10cM - CD34B - 10cM - CD34A and/or Act-6 - 4cM - CD72; CD32A - 11cM - cD3 - 3cM - CD8; chromosome 11: Sod1 and/or Act5 - 4cM - CD29D - 4cM - CD18 - 10cM - CD25B - 16cM - CD17; chromosome 12: CD21B - 1cM - 6Pgdh-2 and/or CD27 - 10cM - CD4B and/or CD16D and/or CD22 - 2cM - CD6A - 3cM - Pgi-1 - 38cM - CD19 - 2cM - Aco-1 - 2cM - Cab-5 and/or 3-48; CD2

Claim 11

The map of claim 7 in combination with at least one of the cDNA clones named therein.

Claim 12

A method for linking an observable characteristic of a tomato plant with a cDNA marker clone of claim 1 comprising comparing the percent recombination of the observable characteristic with the cDNA marker clones of claim 1 and designating the cDNA marker clone having the lowest percent recombination with the observable characteristic as being linked with the observable characteristic.

Claim 13

The method of claim 9 wherein the cDNA clones displaying less than 30% recombination with the observable characteristic are designated as linked with said characteristic.

Claim 14

A method for identifying a substitute DNA marker comprising probing a DNA library with a cDNA marker of claim 1 to identify library DNA substantially homologous to said cDNA marker, observing the recombination rate of said library DNA or a fragment thereof with said cDNA marker or an observable characteristic linked thereto, and designating library DNA having a 0-10% recombination rate as a substitute DNA marker.

Claim 15

A method for determining an heritable association between an observable characteristic of a tomato plant and a cDNA clone of claim 1 comprising: (a) selecting a tomato plant having the observable characteristic and another tomato plant not having the observable characteristic as parents for crossbreeding; (b) extracting DNA from the parent plants and subjecting said DNA to treatment with a restriction enzyme to obtain DNA fragments; separating the fragments from each plant on a neutral gel by electrophoresis and transferring the electrophoresed fragments to a solid support to obtain a hybridization matrix for each plant; (c) hybridizing the matrices of step (b) with a cDNA clone selected from the cDNA clones of claim 1 marking a chromosome or unassigned linkage group which might contain DNA corresponding to said observable characteristic; (d) comparing the hybridization pattern from each parent to determine the presence of polymorphisms; (e) repeating steps (b) - (d) utilizing at least one additional cDNA clone selected from those marking other chromosomes which might contain DNA corresponding to the said observable characteristic and/or using additional restriction enzymes as necessary until polymorphisms have been identified by which the parents may be distinguished from each other by at least one difference on each chromosome or unassigned linkage group which might contain DNA corresponding to the said observable characteristic; (f) crossing the parent plants to obtain an F1 generation and selfing or backcrossing the F1 generation to obtain a segregating generation containing individuals having the observable characteristic; (g) selecting and scoring a statistically significant number of segregating individuals for the percent presence of the observable characteristic; (h) extracting DNA from the segregating individuals of step (g) and following the procedures of steps (b) through (e) to obtain hybridized matrices for each individual showing polymorphisms when compared to the matrices of the parent not having the observable characteristic; (i) identifying cDNA clones which are located within 10 map units (10 cM) of genes conferring the observable characteristic; (j) if no such cDNA clones are identified, identifying cDNA clones which are located less than 30 map units (cM) from genes controlling the observable characteristic; (k) selecting additional cDNA clones from the cDNA clones of claim 1, said cDNA clones having map positions spaced less than 30 map units (cM) from the cDNA clones of step (j), and hybridizing said clones with the segregating generation DNA of step (h); (l) identifying cDNA clones which are located within 10 map units (10cM) from genes controlling the observable characteristic; (m) if no such cDNA clones are identified, selecting additional cDNA clones from the cDNA clones of claim 1, said cDNA clones having map positions spaced closer to previously tested cDNA clones having high percent identity than those having a lower percent identity, hybridizing said clones with the DNA of segregating individuals of step (h) and identifying cDNA clones which are located less than 10 map units from genes controlling the observable characteristic. (n) if no such cDNA clones are identified, repeating step (m) until such clones have been identified.

Claim 16

The method of claim 9 where additional markers are utilized in addition to said cDNA clones and the presence or occurrence of each isozyme occurring in the parent having the observable characteristic is correlated with the percent presence of the observable characteristic in the segregating generation individuals.

Claim 17

The method of claim 9 including locating DNA controlling the observable characteristic on a chromosome map.

Claim 18

A method for screening a tomato plant for the presence of an observable characteristic comprising: (a) linking the observable characteristic with a marker selected from the cDNA clones of claim 1; and (b) testing the DNA of the tomato plant for the presence of RFLP's characteristic of the selected marker.

Claim 19

A method for transferring a desired characteristic from a tomato plant into a second tomato variety comprising: (a) linking the desired characteristic with a previously mapped marker; (b) probing DNA of the second tomato variety with cDNA of claim 1 to determine its DNA profile; (c) crossbreeding a tomato plant having the desired characteristic with a plant of the second tomato variety to obtain an F1 generation; (d) screening F1 individuals for being homozygous for the desired characteristic using the linked marker; (e) probing the DNA of the F1 individuals having the desired characteristic with cDNA of claim 1 to determine their DNA profiles; (f) selecting F1 individuals having the desired characteristic and otherwise having DNA profiles most similar to that of the parent of the second tomato variety for breeding; (g) backcrossing the selected individuals of step (f) with a parent of the second tomato variety to produce an BC1 generation; (h) screening BC1 individuals for the desired characteristic using the linked marker; (i) probing the DNA of the BC1 individuals having the desired characteristic with cDNA of claim 1 to determine their DNA ]profiles; (j) selecting BC1 individuals having the desired characteristic and otherwise having DNA profiles similar to that of the parent of the second tomato variety; and (k) repeating steps (g) - (i) with BC2 and subsequent generations until individuals homozygous for the desired characteristic and having the desired degree of similarity to the parent of the second tomato variety are produced.