Xinlun Liu

Genetic analysis of wheat (Triticum aestivum L.) and related species with SSR markers

Genetic diversity among 19 Triticum aestivum accessions and 73 accessions of closely related species was analyzed using simple sequence repeat (SSR) markers. Forty-four out of 497 SSR markers were polymorphic. In total 274 alleles were detected (mean 6.32 alleles per locus). The polymorphic information content (PIC) of the loci ranged from 0.3589 to 0.8854 (mean 0.7538). The D genome contained the highest mean number of alleles (6.32) followed by the A and B genomes (6.13 and 5.94, respectively). The correlation between PIC and allele number was significant in all genome groups (0.7540, 0.7361 and 0.7482 for A, B and D genomes, respectively). Among the seven homologous chromosome groups, genetic diversity was lowest in group 7 and highest in group 5. In cluster and principal component analyses, all accessions grouped according to their genomes were consistent with their taxonomic classification. Accessions with the A and D genomes were clustered into two distinct groups, and AABB accessions showed abundant genetic diversity and a close relationship. Triticum durum and T. turgidum were clustered together, consistent with their morphological similarity. Cluster analysis indicated emmer is closely related to hexaploid wheat. Compared with common wheat, higher genetic variation was detected in spelt, T. aestivum subsp. yunnanense and subsp. tibetanum. In addition, a close genetic relationship between T. polonicum and T. macha was observed. The results of the clustering and principal component analyses were essentially consistent, but the latter method more explicitly displayed the relationships among wheat and closely related species.

Comparison of the genetic diversity between Triticum aestivum ssp. tibetanum Shao and Tibetan wheat landraces (Triticum aestivum L.) by using intron-splice junction primers

In order to evaluate and compare the germplasm resources of wheat in Tibet, we analyzed the genetic diversity of 136 Triticum aestivum ssp. tibetanum Shao and 119 Tibetan wheat landraces (Triticum aestivum L.) by using Intron-Splice Junction (ISJ) primers. The results showed that polymorphism of PCR products were obtained by 33 primer combinations, which accounted for 11% of the 300 primer combinations produced by 26 ISJ primers. A total of 333 stable bands can be amplified from the T. aestivum ssp. tibetanum Shao and 243 bands were polymorphic, which accounted for 72.9% of the total bands. Tibetan wheat Landraces produced 316 stable bands, of which 197 bands were polymorphic. The polymorphic bands accounted for 62.34% of the total bands produced from Tibetan wheat landraces. The genetic diversity of T. aestivum ssp. tibetanum Shao was higher than that of Tibetan wheat landraces in Tibet, suggesting that T. aestivum ssp. tibetanum Shao can be used as important genetic resource for the breeding and genetic improvement of wheat in Tibet. Matrix (1, 0) was generated according to the presence or absence of the bands produced from a particular wheat accession. Clustering and principle coordinates analysis showed that T. aestivum ssp. tibetanum Shao and Tibetan wheat landraces were divided into two groups. We conclude that high polymorphisms produced by ISJ primers can reflect the genetic diversity between T. aestivum ssp. tibetanum Shao and Tibetan wheat landraces.

Development and discrimination of 12 double ditelosomics in tetraploid wheat cultivar DR147

As an important group in Triticum, tetraploid wheat plays a significant role in the research of wheat evolution. Several complete aneuploid sets of common wheat have provided valuable tools for genetic and breeding studies, while similar aneuploids of tetraploid wheat are still not well developed. Here, 12 double ditelosomics developed in Triticum turgidum L. var. durum cultivar DR147 (excluding dDT2B and dDT3A) were reported. Hybrids between DR147 and the original double-ditelosomic dDT2B of Langdon lost vigor and died prematurely after the three-leaf stage; therefore, the dDT2B line was not obtained. The cytogenetic behaviors and phenotypic characteristics of each line were detailedly described. To distinguish the entire chromosome complement of tetraploid wheat, the DR147 karyotype was established by fluorescence in situ hybridization (FISH), using the Aegilops tauschii clone pAsl and the barley clone pHvG38 as probes. FISH using a cereal-specific centromere repeat (6C6) probe suggested that all the lines possessed four telosomes, except for 4AS of double-ditelosomic dDT4A, which carried a small segment of the long arm. On the basis of the idiogram of DR147, these lines were successfully discriminated by FISH using the probes pAsl and pHvG38 and were then accurately designated.

Molecular cytogenetics identification of a wheat – Leymus mollis double disomic addition line with stripe rust resistance

Leymus mollis (Trin.) Pilg. (2n = 4x = 28, NsNsXmXm) possesses a number of valuable genes against biotic and abiotic stress, which could be transferred into common wheat background for wheat improvement. In the present study, we determined the karyotypic constitution of a wheat - L. mollis double disomic addition line, M11003-4-4-1-1, selected from the F5 progeny of a stable wheat - L. mollis derivative M39 (2n = 56) × Triticum aestivum cultivar 7182, by morphological and cytogenetic identification, GISH (genomic in situ hybridization), FISH (fluorescent in situ hybridization), molecular markers analysis, and stripe rust resistance evaluation. Cytological studies demonstrated that M11003-4-4-1-1 had a chromosome karyotype of 2n = 46 with 23 bivalents, while GISH and FISH analysis indicated that this line contained 42 common wheat chromosomes and two pairs of L. mollis chromosomes. DNA markers showed that the alien chromosomes from L. mollis belonged to homoeologous groups 5 and 6. Evaluation of the agronomic traits revealed that M11003-4-4-1-1 was resistant to stripe rust at the adult stage, while the plant height was reduced and the 1000-grain weight was increased significantly. Therefore, the new line M11003-4-4-1-1 could be exploited as an important bridge material in chromosome engineering and wheat breeding.

Molecular cytogenetic identification of a wheat-rye 1R addition line with multiple spikelets and resistance to powdery mildew

Alien addition lines are important for transferring useful genes from alien species into common wheat. Rye is an important and valuable gene resource for improving wheat disease resistance, yield, and environment adaptation. A new wheat-rye addition line, N9436B, was developed from the progeny of the cross of common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) cultivar Shaanmai 611 and rye (Secale cereal L., 2n = 2x = 14, RR) accession Austrian rye. We characterized this new line by cytology, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), molecular markers, and disease resistance screening. N9436B was stable in morphology and cytology, with a chromosome composition of 2n = 42 + 2t = 22II. GISH investigations showed that this line contained two rye chromosomes. GISH, FISH, and molecular maker identification suggested that the introduced R chromosome and the missing wheat chromosome arms were 1R chromosome and 2DL chromosome arm, respectively. N9436B exhibited 30-37 spikelets per spike and a high level of resistance to powdery mildew (Blumeria graminis f. sp. tritici, Bgt) isolate E09 at the seedling stage. N9436B was cytologically stable, had the trait of multiple spikelets, and was resistant to powdery mildew; this line should thus be useful in wheat improvement.

Molecular cytogenetic identification of a wheat – Thinopyrum ponticum substitution line with stripe rust resistance

Thinopyrum ponticum (Th. ponticum) (2n = 10x = 70) is an important breeding material with excellent resistance and stress tolerance. In this study, we characterized the derivative line CH1113-B13-1-1-2-1 (CH1113-B13) through cytological, morphological, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), expressed sequence tag (EST), and PCR-based landmark unique gene (PLUG) marker analysis. The GISH analysis revealed that CH1113-B13 contained 20 pairs of common wheat chromosomes and one pair of JSt genomic chromosomes. Linkage analysis of Th. ponticum using seven EST and seven PLUG markers indicated that the pair of alien chromosomes belonged to the seventh homeologous group. Nulli-tetrasomic and FISH analysis revealed that wheat 7B chromosomes were absent in CH1113-B13; thus, CH1113-B13 was identified as a 7JSt (7B) substitution line. Finally, adult-stage CH1113-B13 exhibited immunity to wheat stripe rust. This substitution line is therefore a promising germplasm resource for wheat breeding.

Identification of Sitobion avenae F. resistance and genetic diversity of wheat landraces from Qinling Mountains, China

The aphid Sitobion avenae F. is one of the most harmful pests of wheat growth in the world. A primary field screening test was carried out to evaluate the S. avenae resistance of 527 wheat landraces from Shaanxi. The results indicated that 25 accessions (4.74%) were resistant to S. avenae in the three consecutive seasons, of which accession S849 was highly resistant, and seven accessions were moderately resistant. The majority of S. avenae resistant accessions come from Qinling Mountains. Then, the genetic variability of a set of 33 accessions (25 S. avenae resistant and 8 S. avenae susceptible) originating from Qinling Mountains have been assessed by 20 morphological traits and 99 simple sequence repeat markers (SSRs). Morphological traits and SSRs displayed a high level of genetic diversity within 33 accessions. The clustering of the accessions based on morphological traits and SSR markers showed significant discrepancy according to the geographical distribution, resistance to S. avenae and species of accessions. The highly and moderately resistant landrace accessions were collected from the middle and the east part of Qinling Mountains with similar morphology characters, for example slender leaves with wax, lower leaf area, and high ear density. These S. avenae resistant landraces can be used in wheat aphid resistance breeding as valuable resources.

Molecular cytogenetic identification of a wheat-Thinopyrum ponticum substitution line with stripe rust resistance

Thinopyrum ponticum (2n = 10x = 70) has donated rust resistance genes to protect wheat from this fungal disease. In the present study, the line ES-7, derived from the progeny of the crosses between common wheat cultivar Abbondanza and Triticum aestivum—Th. ponticum partial amphiploid line Xiaoyan784, was characterized by cytological, fluorescence in situ hybridization (FISH), genomic in situ hybridization (GISH) and EST-STS marker techniques. Cytological observations revealed that the configuration of ES-7 was 2n = 42 = 21 II. GISH and FISH results showed that ES-7 had two St chromosomes and lacked 5A chromosomes compared to common wheat. The 4A chromosome of ES-7 had small alterations from common wheat. Two EST-SSR markers BE482522 and BG262826, specific to Th. ponticum and tetraploid Pseudoroegneria spicata (2n = 4x = 28), locate on the homoeologous group 5 chromosomes of wheat, could amplify polymorphic bands in ES-7. It was suggested that the introduced St chromosomes belonged to homoeologous group 5, that is, ES-7 was a 5St (5A) disomic substitution line. Furthermore, ES-7 showed highly resistance to mixed stripe rust races of CYR32 and CYR33 in adult stages, which was possibly inherited from Th. ponticum. Thus, ES-7 can be used for wheat stripe rust resistance breeding program.

Molecular cytogenetic identification of a wheat–Aegilops geniculata Roth 7Mg disomic addition line with powdery mildew resistance

Aegilops geniculata Roth is an important germplasm resource for the transfer of beneficial genes into common wheat (Triticum aestivum L.). A new disomic addition line NA0973-5-4-1-2-9-1 was developed from the BC1F6 progeny of the cross wheat cv. Chinese Spring (CS)/Ae. geniculata SY159//CS. We characterized this new line by morphological and cytogenetic identification, analysis of functional molecular markers, genomic in situ hybridization (GISH), fluorescence in situ hybridization (FISH), and disease resistance evaluation. Cytological observations suggested that NA0973-5-4-1-2-9-1 contained 44 chromosomes and formed 22 bivalents at meiotic metaphase I. The GISH investigations showed that the line contained 42 wheat chromosomes and a pair of Ae. geniculata chromosomes. EST-STS multiple loci markers and PLUG (PCR-based landmark unique gene) markers confirmed that the introduced Ae. geniculata chromosomes belonged to homoeologous group 7. FISH identification suggested that NA0973-5-4-1-2-9-1 possessed an additional pair of 7Mg chromosomes, and at the same time, there were structural differences in a pair of 6D chromosomes between NA0973-5-4-1-2-9-1 and TA7661 (CS-AEGEN DA 7Mg). After inoculation with powdery mildew (Blumeria graminis f. sp. tritici, Bgt) isolates E09, NA0973-5-4-1-2-9-1 exhibited a powdery mildew resistance infection type different from that of TA7661, and we conclude that the powdery mildew resistance of NA0973-5-4-1-2-9-1 originated from its parent Ae. geniculata SY159. Therefore, NA0973-5-4-1-2-9-1 can be used as a donor source for introducing novel disease resistance genes into wheat during breeding programs with the assistance of molecular and cytogenetic markers.