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Projecte llegit

Títol: Genotypic characterization of mutant tomato plants for ALCOBAÇA and SELF-PRUNING genes

Estudiant que ha llegit aquest projecte:

Tutor/Cotutor: ROIG VILLANOVA, IRMA

Departament: DEAB

Títol: Genotypic characterization of mutant tomato plants for ALCOBAÇA and SELF-PRUNING genes

Data inici oferta: 16-12-2022      Data finalització oferta: 16-07-2023

Estudis d'assignació del projecte:
    GR ENG SIS BIOLÒGICS
    GR ENG SIS BIOLÒG 23

Lloc de realització:
EEABB

Paraules clau:
Tomàquet, mutant, SNP, genotipat, PCR

Descripció del contingut i pla d'activitats:
El punt de partida del TFG és un creuament entre dues plantes
parentals de tomàquet, cadascuna portadora d'una d'aquestes dues
mutacions: alcobaça (alc) i 'self-prunning' (sp). Aquest
creuament és l'inici d'un programa de millora en aquesta espècie.
Totes dues mutacions són del tipus Short Nucleotide Polymorphim o
SNP, és a dir, causades per un sol canvi de nucleòtid.
Un dels objectius és clonar i seqüenciar el gen SP en plantes
silvestres i mutants per tal d'identificar la mutació.
El segon objectiu és comparar aquesta mutació amb altres
mutacions sp descrites en la literatura.
El tercer objectiu és posar a punt el genotipat dels mutants
mitjançant l'ús d'una modificació de la reacció en cadena de la
polimerasa anomenada treta-primer ARMS-PCR.

Overview (resum en anglès): The tomato has a huge agronomic interest, as well as being consumed all over the world. In this thesis, self-pruning (sp) and alcobaça (alc) mutations will be studied in two varieties of tomato (¿penjar¿ or LC215 and industrial or LC854). The "penjar" tomato is a double mutant for the sp and alc genes, while the industrial tomato is a sp mutant. Prior to this work, these two varieties were used as parentals in a breeding program. They were crossed and their offspring generation F1 was obtained.
The first objective of this thesis was to study whether the sp mutation that both parents have is the same. To address this, genomic DNA was extracted from both parents and the SP gene from each of them was specifically amplified by PCR. The amplified genes were cloned into a vector, sequenced, and their sequence was compared with the wild type available in the databases. The results have allowed us to conclude that, the mutation that causes the sp phenotype in both parental varieties is the same: a substitution from C to T that causes a change of proline (Pro) to a change of leucine (Leu) at position 76 of the protein sequence. Therefore, the F1 generation already has the sp mutation fixed in homozygosity.
The second objective was to develop two protocols based on a PCR modification called tetra-primer ARMS-PCR to genotype the SNPs responsible for the alc and sp mutations in the LC215 and LC854 varieties and their progeny and use them in the breeding program to determine whether the plants of the different generations are wild homozygotes, mutant homozygotes or heterozygotes for each of the mutations. We could not optimize the genotyping of sp but it was optimized of alc. Since in this specific breeding program the sp mutation does not segregate, our alc genotyping protocol will allow progress in the selection of plants for this breeding program.


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