Hirosaki University, Japan.

Sponsored by Prof. Dr.  Ryuji Ishikawa, Hirosaki University

ABSTRACT

The main purpose is characterization of Vietnamese wild rice to apply them as valuable genetic resources to improve cultivars. In the past, one of wild rice accessions has been used to breed superior variety AS996 which are cultivated in Vietnam and getting more cultivating areas. This thesis is composed from two parts, 1) Evaluation genetic resources in the Mekong delta, 2) Characterization of superior variety AS996 bred with a particular wild rice in Vietnam. 1) Evaluation genetic resources in the Mekong delta The wild rice species Oryza rufipogon Griff. is a common perennial known to have been the progenitor of the Asian cultivated rice species, O. sativa L. (Oka 1988, Vaughan 1994). This species serves as a precious reservoir of genetic diversity that can be used to improve cultivated rice with many valuable genes, particularly those conferring resistance to major biotic and abiotic stressors (Brar and Khush 1997, Ram et al. 2007, Xiao et al. 1996, Yuan, Virmani, Mao 1989). Several useful genes have been detected and successfully introgressed from O. rufipogon to cultivars, such as resistance to bacterial leaf blight (BB), brown plant hopper (BPH) and tungro virus, and tolerance to aluminum, sulfate soil, and so on. Despite these advantages of natural populations, wild rice is under serious threat and facing extinction due to ecological changes and human disturbance. Still we can find natural populations in Southern Vietnam. Wild rice accessions were collected from four target sites in the Mekong Delta during 2010 to 2015, in total 166 accessions were collected and compared with control population collected from South-Asian countries as core collection. In addition to these, one hundred wild stocks preserved at Cuu Long Rice Research Institute (CLRRI) were also applied in order to trace mitochondrial deletions. As we have collected many wild rice, we characterized their DNA genotypes by 20 SSR markers and also chloroplast INDEL (cpINDEL) makers. cpINDEL markers were developed from NGS data of wild rice. When we compared to control population from various Asian countries, Vietnamese wild rice showed high diversity within country. Nuclear SSR markers were used to clarify genetic relationships among wild rice populations along and across the Mekong River. A total of 146 alleles over the 20 SSR markers examined were scored among 166 individuals of 24 subpopulations. The expected heterozygosity (He) over the 20 loci examined ranged from 0.067 to 0.764. He ranged from 0.285 in the Can Tho population to 0.659 in the Dong Thap population. Plastid types were defined by INDEL types using eight cpINDELs. From previous data, 14 plastid types were identified as combinations of eight chloroplast INDEL markers. The core collection and the Thai population comprised six and twelve of the 14 plastid types, respectively. Four populations from Vietnam were composed of various plastid types. Only the Can Tho population was composed of a single plastid type, Type 15, which was found in all four populations. Types 16 and 17 were found in the Dong Thap population; Types 18 and 19 were present only in the Intermediate population. Mitochondrial deletions were also applied to evaluate the maternal lineages. Based on a resequencing data of mitochondrial genome by using pair-end reads obtained from a particular Vietnamese wild rice accession in Can Tho, two deletions were presumed. One 699-bp deletion spanned the region from 328,592 to 329,291 bp of the Nipponbare mitochondrial genome. The unique deletion was examined among populations. It suggested that a part of wild rice lineage in Dong Thap province in an upstream of the Mekong river migrated to Can Tho area. These results proved that wild rice populations in Can Tho have unique nature. In addition to such traits, they are known to carry low seed fertility. Cytoplasmic male sterility was once presumed. Thus, candidate mitochondrial chimeric genes were screened with accessions collected in Can Tho. Multiple CMS causal DNA variations were screened by PCR. Then, positive PCR products were found in several cases. These PCR amplified fragments were sequenced for BT type. BT has been reported in wild population but not for HL type. Vietnamese wild rice carried novel chimeric sequences. As we havealready detected complete sterile plants as pollen fertility and aberrant pollen tube growth in particular plants collected at the fields, we speculated that wild rice individuals showing such sterility may have some CMS cytoplasm and may not have fertile restorer gene in order to prefer vegetative propagation. Another interpretation is that they may have altered resources allocation from normal rice plants. Can Tho population prefers vegetative propagation than other populations. It is probably due to constant water level maintained in Can Tho, which is not fit for seed propagation. To allow them to survive, alternative resources allocation to vegetative organs might be selected with unknown genetic mechanism including CMS. 2) Characterization of superior variety AS996 bred with a particular wild rice in Vietnam. AS996 was bred by backcrossing Vietnamese wild rice with improved rice (IR64), they are nearly near isogenic lines sharing almost same genomes except for particular chromosomal segments. AS996 represented high tolerance to poor soil condition as known as low phosphorous soil. Thus, NGS was applied to IR64 and AS996 to compare their genome components as SNPs. Several chromosomal segments showed different SNP numbers between IR64 and AS996. In the most of the segments, AS996 carried higher number of SNPs including chromosome 12. As the chromosome has been reported to carry a particular gene, PSTOL1 for low P tolerance, we tried to amplify the unique gene fragment. However, PCR was failed with AS996 DNA but it was succedded with Kasalath DNA which carries the gene. SNP frequency was higher in the upstream but not the location around the locus. These results suggested that AS996 carries different mechanism to be tolerant to low P soil. We used the information to develop DNA markers for QTL analysis to find linkage relations between markers and the tolerance. We hope the information will lead us further research plan to characterized AS996.