Haplotypes And Phenotypic Association Analysis Of Vernalization And Photoperiod Genes In Chinese Barley

Germplasm resources are the material basis for barley breeding, which is rich in abundance of allelic variations referring to target traits. By taking advantages of abundance allelic variations in germplasms, the candidate gene association analysis is an effective tool in identifying and mining the relevant functional genes and their elite alleles, which provide not only the excellent parent materials with superior goal trait gene alleles but also the molecular markers for assisted selection to barley breeders.Heading date is closely linked with agronomic performance, which is responsible for the regional and seasonal adaptation of barley varieties. Heading at the appropriate time of the year, is not only essential for the completion of barley life cycle, but also a premise to achieve high-yield. Vernalization genes HvVRN1, HvVRN2, HvVRN3 and photoperiod genes PPD-H1, PPD-H2 are responding to the seasonal growth habit and flowering time in barley. In this study, a set of Chinese barley germplasm was used for maker based genotyping, gene re-sequencing and allelic variation analysis of these genes. In the meantime, the genome-wide association analysis was performed based on the SNP array genotyping and multi-ecological environment phenotyping. The main results are summarized as follows:1. The proportions of dominant and recessive alleles of HvVRN1 were 54% and 46% respectively in China’s cultivated barley. But no recessive allelic gene was found in China’s wild barley. Dominant allele V2 of HvVRN2 is main type and account for 86%, comparing with a 14% proportion of recessive allele v2 in Chinese barley. The frequency of v2 is only 3% and 10% respectively in Chinese wild barley and landrace, but much higher in modern variety, accounting for 60%. v2 distributed mainly in high latitude northern spring barley regions. And among all the 12 barley ecological zones of China the frequencies of v2 reduced with latitude declining. V2 distributed all in the southern winter barley regions except for south-western plateau zone.2. Out of the ten known HvVRN1 haplotypes, except for HvVRN1-7(V1-7) nine were found in China’s barley gerplasm core collection. Haplotypes V1-2 and V1-3 were restricted to modern cultivars and were deduced to introduce from foreign barley by pedigree tracing. Haplotypes V1-10 was found only in the barley landraces from the most southern subtropical districts of China and without in the barleys from any other countries of the world. Therefore V1-10 was concluded as originated in China. The frequencies of the nine haplotyps of dominant V1 were as follows: V1-8(35%) > V1-9(7%) > V1-5(6%) > V1-4(4%) > V1-1(3%) > V1-6(3%) > V1-10(2%) > V1-2(0.6%) > V1-3(0.3%) in China’s barley. Haplotype diversity of HvVRN1 was higher in the cultivated than in wild barley. There were only 2 haplotypes in the wild barley, conspicuously comparing with 8 and 9 haplotypes in the landraces and modern varieties respectively. Out of the ten allelic variations of V1, the haplotype V1-8 occurred at the highest frequency in China’s barley and mainly distributed in the Qinghai-Tibet plateau area. V1-1 and V1-5 distributed in northern spring barley regions; especially in the barley zone with a latitude >44°N only V1-5 was found. v1 and V1-6 were carried mainly by winter barley and mostly distributed in southern winter barely regions with a latitude 27°N-37°N. V1-9 concentrated in the south-west winter barley zone, and V1-10 is distributed in the most southern region with the lowest latitude(<25°N) in China. A total of 20 multi-locus haplotypes of HvVRN1/HvVRN2(V1/V2) were identified and the geographic distributions of V1/V2 were same with HvVRN1.3. Seasonal growth habit was identified under multi-ecological conditions for 703 Chinese barley accessions. Morphological association analysis of allelic variations confirmed that barley growth habit was determined firstly by the gene HvVRN1. The “0.44 kb core region” in the first intron of HvVRN1 is directly associated with vernalization sensitivity. Haplotypes with deletion of the 0.44 kb including V1-1, V1-2, V1-3, V1-5 and V1-10 all showed a strong spring growth habit regardless of HvVRN2 genotype. Haplotypes v1, V1-4, V1-6, V1-8 and V1-9 containing the 0.44 kb region but with deletions in other region of the first intron showed all three types of growing habit: spring, facultative and winter. In them, the reduction length resulted from fragment deletion in the first intron of HvVRN1 was relative to the sensitivity to low temperature, because the larger of the deletion fragment, the higher of the spring growth habit frequency.4. Genome-wide association study demonstrated that HvVRN1(5H) and HvVRN2(4H) were main locus related to barley growth habit. HvCEN on the 2H chromosome had minimal effect on growth habit. However, HvVRN3 on the 7H chromosome had no effect on growth habit in this study. Some other loci on 1H, 2H and 6H were found to associate with seasonal growth habit in addition to the previously reported sites.5. Genome-wide association study for photoperiod sensitivity in 281 Chinese spring barley accessions showed that PPD-H1 was a major locus affecting heading date under long day condition. Especially in the Haerbin environment(>44°N) with the longest day light during barley growing season, it had the most phenotypic interpretation ratio of the heading date. But under fall-sown and short day condition in low latitude eco-regions PPD-H1 did not play a role in barley heading. In general, more loci associated with heading date were found on the 2H than on any other chromosomes. The region of 53.47-58.78 cM on 2H chromosome was associated with heading date in multi-environments.6. In 377 Chinese barley accessions, 67 allelic variations were found in HvVrn3 including 61 SNPs and 6 InDels. And all of them distributed in promoter except for that 4 of them were present in intron. The alleles of recessive Hvvrn3(v3) and dominant HvVrn3(V3) accounted for 97.1% and 2.9% in Chinese barley respectively, classified according to the promoter SNP927(T/C) and intron I(TC-AG) variations. These 67 allelic variations constituted 10 haplotypes. Among them H1 and H9 mainly distributed in Qinghai-Tibet Plateau and H1 is restrict in this region. H4 was distributed in high latitude areas and H2 and H7 had broad geographic distributions. H4 performance late and H8 did early flowering in phenotyping in multi-environments. The difference between these two haplotypes were 4-15 days in heading date, therefore H8 have potential value in barley improvement for early varieties. Three haplotypes in promoter and intron I were investigated according to functional variations. Among them T-0-4-AG was 4-14 days earlier than C-0-4-TC and C-4-0-TC in heading date.7. Surveying the distribution of PPD-H1 allelic polymorphism across 381 barley germplasm accessions, forty-four variations were discovered, including 5 InDels and 39 SNPs. Out of the 39 SNPs 12 resulted in amino acid changes. These 44 allelic variations constituted 17 haplotypes in all. Out of the major haplotypes, H2 and H12 concentrated in Qinghai-Tibet plateau area, H4 mainly distributed in high latitude, H5, H6, and H15 have broad geographic distribution. The Ppd-H1 and ppd-H1 accounted for 87.9% and 12.1% respectively in Chinese barely germplasms.8. One of SNP in the PPD-H1 CCT-domain, the SNP37 was associated with barley heading date in long day condition by mixed liner model(MLM) analysis. Haplotypes H5, H6 and H15 were earlier 4-14 days than H2, H4 and H12 in Qinghai, Beijing and Haerbin. Although there was a highly significant difference in heading date between Ppd-H1 and ppd-H1 in long day condition, no difference existed between them in short day and fall-sown condition.9. Thirteen variations including 11 SNPs and 2 InDels of PPD-H2 was detected in 364 Chinese barley accessions. Out of them SNP10(A/G) was associated with heading date in all spring-sown and long day environment but not in fall-sown and short day condition. All the 13 allelic variations of PPD-H2 constituted 9 haplotypes and further were divided into two groups: early flowering group including H2, H3, H4 and H6 and late flowering group including H7, H8 and H9, with a difference of 3-13 days in heading date.