Identification And Development Of Molecular Makers Associated With Sex Determining Gene In Asparagus

Asparagus officinalis L. is a popular and well-welcomed nutritional health-keeping vegetable. The cultivated asparagus(Asparagus officinalis L.), "the king of vegetable", is a dioecious plant. A region called the M-locus, the sex determination gene, located on a pair of homomorphic sex chromosomes L5 controls the sexual dimorphism in asparagus. The male is M-, and the female is mm. According to the fact of experiences, Male is preferred to female in asparagus breeding and cultivation because of its higher yield potential with a longer life span and better tolerance to diseases around the world. So it is proposed that theory of all-male breeding utilizing of heterosis and all-male advantages in the way of genetics breeding, the heredity principle is based on MM×mm-Mm. Early selection of male plants in asparagus is desirable but very difficult since it takes two years from seedling to flowering. Sex-linked molecular markers offer solution to asparagus breeding by early identification of sex at seedling stage. The main results are as follows:1. DNA extracted ways in the genome of asparagus between modified CTAB,SDS,extraction kits ways are compared:the comprehensive result of modified CTAB way is ideal for purity,quality and completeness in cladophylls part of Asparagus; 2% PVP and 0.1% 2-mercapthoethanol could remove polysaccharides,polyphenols residues and prevent be oxidized; based on the results of herphrodites phenomena investigations, choices of high proporational bisexual cultivars self-crossing segregative (female and male) S1 populations with 10 male individual plants and 10 female plants each, are constructing 4 pairs of DNA pools (B23,B24 B25,B33) and 1770(MM) including its offsprings populations G190-1 (mm) totaling 5 pairs, which are used to devise sex-determination genetic markers by BSA (bulked segregational analysis) ways;2. ISSR is used to study the sex genetic markers on Asparagus officinalis L.,the best reactive conditions are analyzed based on the single-factor analysis:in the 25μL reactive volume of ISSR marker technology, genome DNA 50ng,primer 5μmol/L 2μL,dNTP200μmol/L 2uL,10×buffer2.5μL,Mg2+ 25mmol/L2.5μL,Taq enzyme 1U; reactive procedures:94℃de-naturations 5min; 32 cycles (94℃de-naturations lmin, 48℃～53℃annealing 1min,72℃elongation 1min); final elongation 72℃10min;3. In the analysis of 54 selective ISSR primers,2 polymorphic fragments are detected with ISSR3 in 5 pairs of DNA pools, named Asp₁ and Asp₂, respectively. Then the fragments of Asp₁ and Asp₂ are recovered, cloned and sequenced. Analysis of the sequence shows Asp₁ with 778bp is abundant in AT and contains 4 shorter open reading frames and Asp₂ with 993bp is also abundant in AT and contains 6 shorter open reading frames. After blastn analysis in NCBI, they are all specific fragments in the genome of Asparagus.4. The fragments of Asp₁ and Asp₂ are linked close to sex determination gene after individual DNA test. Dependence of sequencing results, specific primers are designed to transform the ISSR3 marker to more stable SCAR markers, which are namedSCARl(F:5'-ATTGCGAGCATTGTAATCTTCTTC-3',R:5'-TTGTCATTGG ATAGGGAGT-3'),SCAR2(F:5'-TCATTGGATAGGGTTGGAGTCGG-3',R:5'-TG TGCATTTCATCACCTCGGTCT-3') based on Asp₁. SCAR1 is a co-dominant marker with linkage with sex determination gene. The sex-linked co-dominant SCAR 1 marker is obtained, which would be efficient to identify different sexes of Asparagus and play an important role in mapping sex determination gene and assist all-male breeding in asparagus, primary discrimination efficiency is 83% after individual test; while SCAR2 is only a specific marker of Asparagus with no polymorphisms in sex determination gene. And SCAR3 is devised dependence on Asp₂ sequence. This marker can stably amplify on the genomes of five pairs of DNA pools and 12 individual plants, which is only present on the male plant DNA of asparagus. So this marker is linked with sex determination gene on asparagus with 83% of primary discrimination efficiency after individual test.5. SRAP reaction system suitable for asparagus genome is set up utilizing single factor analysis:80ng of template DNA,3.0mmol/L of Mg2+,0.3mmol/L of dNTPs, 0.2μmol/L of forward and reverse primers each and 1U Taq DNA polymerase in 25μL total volume. The reactive program is 3 min of pre-denaturation at 94℃, five cycles of three steps:30 sec of denaturation at 94℃,30 sec of annealing at 35℃and 1.5 min of elongation at 72℃. In the following 35 cycles, the annealing temperature is increased up to 50℃, with a final elongation step for 10 min at 72℃. And the two methods of analysis between agarose gel electrophoresis and poly-acryamide gel electrophoresis are compared, the results show the detection efficiency in 6% polyacryamide gel is higher than agarose gel for SRAP analysis.6. Then this excellent SRAP system is screened with 256 primer pairs and 239 pairs can amplify clear and stable bands siezing 93.36% of the total primers. Extraction of different products amplified by ME3+EM12 is conducted in Cultivar B23 around 200bp-500bp, then 307bp is obtained named Asp-3 after sequencing and blastn analysis shows it is a new and specific fragment of Asparagus. Then primer ME3+EM12 is conversed into SCAR4(F:5'-CCTGTTCCGTTTACTTA GGACAATC-3', R:5'-GGGTATTATTTGGAGAAGGTGGAG-3') based on the sequences, shows no polymorphisms between different sexes in every cultivar.