Development And Validation Of Functional Marker For Resistance To Pre-harvest Sprouting

Pre-harvest sprouting (PHS) is defined as the germination of grains in the ear before harvest, and it causes down-grating of quality, severely limits the end-use application, leading to substantial losses in price and yield. PHS is a serious problem in most parts of the wheat growing regions. The PHS tolerance results from the combined effects of multiple factors influencing spike water uptake and drying, grain dormancy, and rate of degradation of grain components during germination. The major factor involved in the PHS under wet weather condition is grain dormancy. Research into the mechanism of seed dormancy using mutants and transgenic plants has revealed that abscisic acid (ABA) plays a predominant role in dormancy during seed development. Both of ABA sensitivity and ABA content are correlated with the level of dormancy, and CYP707A1 might be a major gene that catalyse the 8'-hydroxylation of ABA to Phaseic acid (PA). In the present study, wheat TaCYP707A1 gene was cloned, and a STS marker for PHS tolerance, Dorm-1, was developed and validated. In addition, 618 wheat cultivars were screened with Dorm-1, and the markers related to photoperiod sensitivity, PPO activity and YP content were used to analyze the 95 spring wheat cultivars from Heilongjiang province.The main results obtained in this study are summarized below.1. The cDNA sequence of barley ABA 8'-hydroxylation gene HvCYP707A1 (GenBank accession AB239299) was used as a probe for BLAST search against the common wheat (Triticum aestivum L.) EST database in GenBank. All the ESTs sharing high similarity with the reference gene were subjected to contig assembly. Primers were designed based on the constructed contigs to clone the wheat CYP707A1 gene, designated as TaCYP707A1. The complete genomic DNA sequence of TaCYP707A1 gene comprised 2,225 bp, with five exons and four introns. The corresponding cDNA sequence of TaCYP707A1 was 1,737 bp, containing an open reading frame (ORF) of 1,431 bp, a 42-bp 5'-untranslated region (UTR) and a 264-bp 3'UTR, with 94.9% sequence identity with HvCYP707A1 gene (AB239299). The phylogenetic tree indicated that the deduced amino acid sequence of TaCYP707A1 gene was highly similar to those of barley and rice. The TaCYP707A1 gene was localized on chromosome 6BL using a set of Chinese Spring nullisomic-tetrasomic lines and ditelosomic line 6BS.2. A co-dominant STS marker related to PHS tolerance was developed and designated Dorm-1. In most cases, the marker can amplify a 606-bp and a 486-bp fragment from the tolerant and the susceptible cultivars, respectively. The STS marker was localized to chromosome 7BL using a set of Chinese Spring nulli-tetrasomic lines and ditelosomic line 7BL. A total of 104 white-grained Chinese wheat cultivars and advanced lines were used to validate the association between the polymorphic fragments of Dorm-1 and PHS tolerance. The results indicated that the 606-bp fragment amplified by Dorm-1 was significantly associated with PHS tolerance, whereas the 464-bp fragment with PHS susceptibility, suggesting that Dorm-1 could be used as an efficient and reliable marker to evaluate wheat germplasms targeting for PHS tolerance. 3. A total of 618 Chinese and international wheat landraces and improved cultivars collected from tewelve Chinese wheat growing zones and ten countries were tested by Dorm-1. The frequency of the 606-bp allele was 8.5% and 7.9% in Chinese and international wheat lines, respectively. Furthermore, the results of detection with Dorm-1 in red kernel cultivars indicated that the marker can also be used to evaluate their PHS.4. A total of 95 cultivars, collected from Heilongjiang province, were tested with markers Ppd-D1, PPO18, PPO16, PPO29 and YP7A, respectively. The results showed that most of the predominant cultivars grown in this province exhibited photosensitive characteristic, which is mainly due to the unique ecological condition of the area. Both of the genotypes associated with higher and lower PPO activity were found, and the proportion of the PPO-2Aa and PPO-2Db controlling high PPO activity were 51.8% and 42.2% respectively. At the same time, the proportion of the allelic variations at Psy-A1 loci controlling high and low YP content were 41.1% and 58.9% respectively. However, most representive cultivars backbone parents , such as longmai 26, longmai 30, longfumai 12, longfumai 16, kehan 18, kehan 19, and kehan 20, present Psy-A1a controlling high YP content, it may due to the lack of selective pressure.