| Garlic (Allium sativum L.), an asexually propagated crop, is grown as an important vegetable and medicinal plant. China is the biggest garlic producer in the world. However, little is known about the genetic background of garlic germplasm resources in China. The characteristics of vegetative propagation of garlic makes it very difficult to construct mapping populations for research on elite genes. With the increasing requirement for nutritional quality, especially for high allicin content, it is urgent to carry out relative research for sustainable development of garlic industry to keep China at the present prosperous position in international garlic trade.In this study, the genetic diversity and the genetic structure of garlic germplasm in China were evaluated based on the morphological traits and three kinds of molecular markers (AFLP,SSR and InDel ); an efficient UPLC detection method for allicin content in garlic bulb was developed and used to detect the allicin content among 212 accessions of garlic germplasm; the gene structure and diversity of alliinase genes from garlic within encoding region were studied by TAIL-PCR and RT-PCR technology; a novel InDel marker was developed according to the sequences of intron 2 of alliinase genes; association mapping method was used to study the relationship between allicin content and InDel marker. The major results are as follows:1. Phenotypic diversity and classification of garlic germplasm in China were studied based on morphological traits by methods of clustering, principal component analysis, and correlation analysis. The results showed that the garlic germplasm in China presented a wide diversity from all traits. The five-grade classification method for each quantitative trait was brought forward. The results of clustering and principle component analysis indicated that clustering factors were mainly from qualitative traits. To avoid the overrating effect from qualitative traits, eight important quantitative traits related to bulb yield were evaluated. Principal component analysis showed the cumulative contribution proportion of the first three components was up to 74.83% and three traits (bulb weight, bulb diameter and clove number) were included in the first components which could be the most important traits affecting bulb yield. Correlation analysis suggested that bulb yield was significantly related with seven of eight characteristics except clove back width, and the most highly related with bulb diameter and bulb weight. All accessions of garlic germplasm were divided into 6 groups according to yield and the analysis of principal coordinates. Moreover, three accessions yielded above 15×103 kg ha-1 were selected out.2. Analysis of population genetic structure and clustering analysis of garlic germplasm was performed based on AFLP, SSR and InDel. Totally, 502 allels were amplified by AFLP, SSR and InDel primers, 492 of them were polymorphic among 212 accessions of garlic. All accessions were divided into 5 groups by both structure analysis and neighbor-joining clustering. However, the Shannon's index of each group assumed by genetic structure analysis was smaller than that assumed by neighbor-joining clustering, which indicated the genetic structure analysis could interpret genetic relationship among the individual accessions in more details. Most of traits including allicin content were slightly affected by population structure, which indicated the germplasm in this study was acceptable to be the populations for association mapping.3. An efficient UPLC method for quantitative determination of allicin in garlic bulb was developed and used to detect the allicin content among 212 accessions of garlic germplasm. In the UPLC method,methanol/water (50:50, v/v) as a mobile phase which ran through at a constant flow rate of 0.3 mL min-1 was performed onUPLCTM BEH C18 column. The stability of allicin extracted with water from garlic bulb powder was evaluated in the following conditions: phosphate buffer , pH value from 1.50 to 11.0; temperatures, from -20 to 85℃, and light in absence or presence. Results indicated that allicin in solution was sensitive to pH and temperature of storage, but not to light. At room temperature, allicin was most stable with pH 5.0 to 6.0, but it degraded quickly at lower or higher pH. but it degraded quickly when the pH was lower than 1.5 or higher than 11.0. Allicin degraded more quickly when temperature was higher than 40℃, and especially when temperature was higher than 70℃. At room temperature (25℃), allicin in water could be stored for 5 days without obvious degradation. Higher the concentrations of allicin in solution were somewhat more stable than low concentrations. Allicin content of garlic germplasm ranging from 0.82 to 3.01% was significantly different. Based on investigation of 52 accessions, the accessions with the higher allicin content were more resistant against the Delia antique Meigen.4. The gene structure and its diversity of alliinase genes from garlic within coding region were studied by TAIL-PCR and RT-PCR technology. Results confirmed that the alliinase genes possess 5 exons and 4 introns. Extraordinary diversity was found among these alliinase genes, especially within introns. Besides, some InDels and couples of candidate SNPs positions were found within exons as well. Combining the introns and exons, 10 types of alliinase genes were predicted. Three garlic germplasm E1, GH1and CN313, from Uzbekistan (Central Asia), USA (West), and China (East) respectively, were used for analyisis of diversity and evolvement of alliinase genes. Cluster tree based on the alliinase genes from genomic sequences supported the idea that garlic was spread from the original center (central Asia) to the west countries and then to the east.5. A novel alliinase gene specific InDel-primer flanking the intron 2 was designed according to the alliinase sequences from the three accessions of garlics. The InDel primer produced 10 polymorphic alleles and divided 212 accessions of garlic into 5 genotype groups. The results indicated that the InDel maker was valuable for the diversity and classification analysis of garlic germplasm.6. The association analysis based on candidate gene method was used to study on the relationship between the InDel marker and allicin content. Results showed the high allicin content was significantly linked to the genotype C with bands of 335 and 327 bp, and low allicin content was significantly linked to genotype I with bands of 343, 340 and 323 bp. The InDel maker could be an assisted marker to select high allicin content variety from garlic germplasm.
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