Study Of Self-incompatibility From Almond In Xinjiang (Amygdalus Communis L.)

Almond is the famous dried fruits and oil trees in the world, which has a higher nutritional and medicinal value. Xinjiang in China is one of 32 almond origins all over the world. Almond is a gametophytic self-incompatible fruit trees, so it must configure pollination varieties or through artificial pollination to ensure high and stable yield in the aspects of production and cultivation. The first to consider is the affinity between varieties when configuring pollination varieties, and affinity between the two varieties depends on their S-genotype, hybrid incompatibility when 5-genotypes is the same, or hybridization affinity. This study take 24 almond cultivars in Xinjiang as materials, taking isolation and identification of the 5-gene and determine of S-genotypes as targets, using field pollination, style cultivated in vitro and 5-allele specific PCR methods, launched the research of molecular biology and the affinity of hybrid. The aims are to provide important scientific basis of almond cultivation and genetic improvement, provide theoretical basis and practical guidance for carrying out a comprehensive self-incompatibility in almond’s another fruit trees. The main results are as follows:1 Field pollination experiment showed that in the five Xinjiang almond varieties, which was ’Zhipi’,’Shuangguo’,’Make’,’Kexi’and’267’,’Shuangguo’showed some self-compatibility and its setting rate of self-flower was 29.05%, setting rate of self-flower of’Zhipi’was 12.76%, those whom of ’Make’,’Kexi’and’267’was less than 5%, which indicated typical self-incompatibility. Under natural conditions, the setting rate of ’Zhipi’was the highest, which reached to 37.53%, setting rate of natural pollination of’Kexi’was the lowest, only 4.3%; Under the conditions of artificial cross-pollination, the fruit setting rates of all varieties were significantly increased, the fruit setting rates of pollination combination of’Shuangguo’x’Zhipi’ was up to 68.09%. When using pollens of different cultivars pollinated to the’Zhipi’, all could obtain a higher setting rate, the fruit setting rate pollinated using pollen of’Make’was the highest, up to 63.43%; When using pollens of different cultivars pollinated to the’267’, which obtained lower setting rates, only the fruit setting rate pollinated using pollen of ’Zhipi’was up to 36.25%, those whom of’Kexi’was only 16.18%.2 In vitro culture of style showed, when the five cultivars of ’Zhipi’,’Shuangguo’,’Make’,’Kexi’ and’267’carried out self-cross, the number of style growing pollen tubes were less, the rate was low. Combination of self-cross of’Shuangguo’ had growed pollen tubes from three style incision, which was 1/3 location,1/2 location and base of style, respectively, which indicated some self-compatibility; Two combination of self-cross of’Zhipi’and’Kexi’had nothing pollen tubes growed from any style incision, another combination of self-cross of’Make’ and’267’had growed pollen tubes only from 1/3 location of style incision, which showed typical self-incompatibility.When the five varieties outcrossed each other, the number of style growing pollen tubes of the most crosses were higher than self-cross, which meaned configuration of major cultivars and pollination tree in the scientific manner could increased effectively the setting rate of almond. When the same male parent and female parent is not the same affinity performance between the different species varied greatly, which showed pollinated varieties had highly selective when the almond in outcrossing. Therefore, we should choose appropriate pollinated varieties in the production, meanwhile, noting that the cultivars and pollination species had a reasonable collocation. When selecting pollinated trees and cultivars, we also should consider economic value of pollinated trees. Three varieties of ’Zhipi’,’Shuangguo’,’Make’could use as pollinated trees each other, their outcrossing affinity were high, they acted as pollinated trees also had higher economic value.3 The test of almond genomic DNA extraction using four methods showed that the DNA quality extracted by improved CTAB method was the best, OD260/OD280 was close to 1.8, and there were no smearing phenomenon in the picture of electrophoresis, which indicated purity of DNA was good, RNA was less, pollution of polysaccharide, protein, phenol and other impurities was less. DNA precipitation was transparent jelly, and without pollution caused by phenolic oxidation or other pigments. The next was CTAB method and improved SDS method; The purity of DNA extracted by using SDS method was very poor, OD260/OD280 was less than 1.8, and there existed pollution of protein or phenol.4 Amplification coefficient and amplification successful rates of different primer combinations to tested almond cultivars vary widely, amplification effect of primer combination of AmyC5R+AS1Ⅱwas the best.5 The amplification results of eight primer combinations showed if we wanted to carry out comprehensive and accurate identification of the S-genotypes from Xinjiang almond varieties, in addition to using different primer combinations, but also need to further develop and design new specific primers according to the 5-gene sequence of Xinjiang almond.6 Using primer combination AmyC5R+AS1Ⅱcarried out S-gene specific PCR to 18 genomic DNA of Xinjiang almond cultivars,32 clear fragments were amplified. After cloning and sequencing, the software of DNAMAN analysis showed the bands located at identical position of different cultivars, their DNA sequence similarity was above 99%. So we could think they were the same gene fragments, that meaned 32 gene fragments total included 11 nucleotide sequence, whose size involved 1688 bp (Snt),1404 bp (S,9),1330 bp (S61),1222 bp (S24),1141 bp (S25),1087 bp (S17),979 bp (S,o),785 bp (S63),777 bp (S6), 690 bp (S50),602 bp (5/5), respectively.7 According to the identification results of S-gene and cross pollination test, we determined S-genotype of 10 almond cultivars, which was ’Amaiti’ (S15S17S63),’Hongbaoshi’(S15S17),’Shazhou’ (S15S17),’Yingzui’(S10S24),’Aifeng’(Sn1S25),’Bianshitou’(Sn1S25),’Taobiantao’(S6Sn1),’Kaixinguo’(S6S61), ’Shuangguo’(S63Sn1) and ’Zhipi’(S50S61).