Chromosome Diversity Of Qingke Landraces In Tibet Based On Tandem Repeat FISH

Tibet is the driest,coldest and windiest region in the same latitude in the world.It has the characteristics of a maritime climate in the surrounding lowlands and plains,forming an extremely complex and diverse plant ecological environment.Qingke,also known as naked barley,is a characteristic food and feed crop on the Qinghai-Tibet Plateau.A lot of work has been done on Qingke landraces genetic research.However,the characteristics of chromosomal variation during the evolution of Qingke landraces are not clear.For the test material,the chromosome composition of the tested varieties was identified by developing a repeat sequence oligonucleotide probe set.The main results obtained are as follows:The second-generation sequencing was used to obtain the barley genome sequence,and the unit and content of the barley tandem repeat sequence were identified through bioinformatics analysis,and four tandem repeat clusters HV_CL34,HV_CL41,HV_CL43,and HV_CL51 with a length of 118-444 bp were identified,based on the published barley repeat sequence pHvA14-2-1～3,a total of 7 oligonucleotide probes were designed.FISH analysis found that these probes can produce clear signals on all chromosomes of barley.Select the probe with the clearest signal,we use HV_CL43 and previously developed probes（GAA）10,（AG）10,（ACT）10,45S rDNA oligonucleotides（Grass-18s-1,Grass-18s-2,Grass18s-3,Grass-5.8s-1,Grass-25s-1,Grass-25s-2,Grass-25s-3,Grass-25s-4,Grass-25s-5,Grass-25s-6）and 5SrDNA oligonucleotides（Grass-5S-1,Grass-5S-2）assembled a new barley oligonucleotide probe multiplex ONPM#8,which can provides a simple,economical and efficient tool for barley chromosome analysis.Using ONPM#8 FISH,the reference karyotype of the purple barley was constructed.All chromosomes have a high similarity with the Qingke landraces,of which 2H has the most abundant banding patterns,followed by 3H,and the least banding is 7H,according to this karyotype,chromosome-specific banding can clearly identify all the barley chromosomes.Based on this karyotype,karyotypes of 137 other local highland barley varieties were constructed,each landrace observed at least 3 individual plant.The karyotype comparison shows that the karyotypes of the tested landraces are highly homogeneous,and no obvious karyotype heterogeneity was found in the tested landraces,but the karyotype differences among different landraces are obvious.Among them,19 polymorphisms were found on 7 chromosomes,of which 3H had the most（5 types）,the others are 6H（4）>2H=4H（3）>1H（2）>5H=7H（1）.statistical analysis shows that the six polymorphic types 5H-1,7H-1,2H-3,4H-2,1H-1 and 6H-4 have a frequency of more than 50%,perhaps they are the dominant types in the region.CPIC analysis found that 3H had the largest variation,5H and 7H had the smallest variation.The karyotypes of the tested landraces are divided into 53 types,of which the 2nd and 9th karyotypes are the most,and the 16 karyotypes contain only one landrace.Based on the basic data published by the China Germplasm Resources Information Network,the characteristics of six different types（ET1,ET2,WT1,WT2,WT3 and NT）of Qingke landraces divided by GBS（Genotyping by sequencing）were compared,where ET means estern Tibet,WT western Tibet,and NT means Tibet landraces were genetically similar to inland genotypes.The plant height,thousand-grain weight,number of spikelets,crude protein and lysine content varied in these types,where significant difference was seen among several groups.In breif,Qingke from eastern Tibet,western Tibet（WT1+WT2+WT3）and NT ones,ET Qingke（ET1+ET2）increased plant height and grains per spike,while WT ones increased thousand kernel weight,content of crude protein and lysine.No significant difference was seen on total starch content among all groups.Among six types of Qingke,the highest plant height and grains per spike was found in ET2 type,while the highest thousand-grain weight and lysine content in WT2.