Screening A Core Set Of Informative SSR Primers For Identificating Cotton Cultivars And Lines(G. Hirsutum L.)

It is very important to identify cultivars rapidly and precisely for plant variety certification, plant varieties protection, fake seeds identification and property right dispute. Thus, it is significantly important to research on the rapid, high-efficient and stable cultivar identification system, SSR molecular makers are more widely used in cultivar identification because of its own advantages. This study aims to select a set of core primer pairs and develop a system for cotton cultivar identification, genetic clustering analysis of the tested materials and authenticity identification of 67 cotton varieties in Shandong provincial regional trial in 2009 were also performed based on DNA fingerprinting database derived from the selected 11 core primer pairs,the main research conclusions are as follows.1. A rapid and stable system applied for the cotton cultivar identification was developed: two different methods of DNA extractiong from cotton leaf and seed are established respectively, touchdown PCR increases specificity and sensitivity in PCR amplification, a sample and rapid method improves the efficiency of silver staining of DNA banding in PAGE gel.2. Genomic DNA from 32 approved and commercial cotton cultivars with larger planting areas were amplified with 2000 primer pairs selected from CMD database, cottonDB database and relevant research papers, 26 core primer pairs with high polymorphism and stability, clear amplified banding were selected, which accounting for 1.3% of primers used in this study and covered 20 cotton chromosomes. Genetic clustering analysis by UPGMA of 32 cotton cultivars was performed based on DNA fingerprinting database derived from the the reducing number of the core primers (26 pairs, 11 pairs, 8 pairs), the results showed that the difference of 32 approved cotton cultivars at DNA level based on 26 and 11 core primer pairs was little, but it was big when the core primer pairs reduced to 8, thus 11 core primer pairs were proposed as preferred primer pairs for identification of cotton cultivars.3.A formula for calculating the maximum number of cotton cultivars that the core primer pairs can identify in theory was advanced: N=G1×...×Gn, G1...Gn, and the probability of the different cultivars having the same DNA fingerprinting was: P=1/N; 11 core primer pairs can identify the maximum number of cotton cultivars in theory is 419904 .The probability of the different cultivars having the same DNA fingerprinting was :2.38×10-6. According to theory, 11 core primer pairs were effective in cotton cultivar identification.4. The results of genetic clustering analysis of 32 cotton cultivars based on DNA fingerprinting database derived from 11 core primer pairs demonstrated the pedigree of them, cultivars with the same parents having a great genetic similarity coefficient, and clustered into the same group of UPGMA dendrogram. Difference of 32 approved cotton cultivars at DNA level was in accordance with the result of pedigree analysis of them.5. The result of authenticity identification of A approved cultivar based on DNA fingerprinting database derived from the 11 core primer pairs showed that A cultivar's fingerprting was exactly the same as Xinqiu 4 cultivar's, the fact is that leaves of A cultivar were from Xinqiu 4 cultivar, the result was in accordance with the fact.6. The deduced result of authenticity identification of 67 cotton varieties in Shandong provincial regional trial in 2009 based on DNA fingerprinting database derived from the selected core primer pairs was that the number 2 and number 15 cotton lines in Shandong provincial regional trial was the Lumianyan 37 and Hanmian 559 cotton cultivar respectively, and the number 38 and number 45 cotton lines in Shandong provincial regional trial was the same cotton lines as well as the number 11 and 46.7. Genetic clustering analysis of different combinations of cotton cultivars (lines) used in this study was performed based on DNA fingerprinting database derived from the 11 core primer pairs, the result showed that genetic basis of the tested materials was limited, genetic basis of cultivars from the same breeding unit is limited, and it is relatively large in different areas and different breeding units.