Genetic characterization of wild barley populations (Hordeum vulgare ssp. spontaneum) from Kazakhstan based on genome wide SNP analysis.

The wild ancestral form of barley, Hordeum vulgare ssp. spontaneum, is a valuable source for gene enrichment of cultivated barley. The purpose of this work was to study the area of distribution as well as the extent and structure of genetic variation of wild barley populations grown in Kazakhstan. It was found that distribution of wild barley populations in Kazakhstan is restricted to the most southern province. A genome wide single nucleotide polymorphism (SNP) analysis was performed in order to study the level of the genetic diversity in 96 accessions representing 14 wild barley populations from Kazakhstan and 25 accessions from the Middle East which is the center of diversity of this subspecies. The oligonucleotide pooled assay was used to genotype 384 SNPs distributed throughout the genome. In total 233 polymorphic SNPs were selected for further statistical analysis. The level of genetic diversity of wild barley populations from Kazakhstan was predictably narrower (He = 0.19 ± 0.01) in comparison with wild barley samples from the Middle East (He = 0.29 ± 0.01). The results suggested that H. vulgare ssp. spontaneum populations in Kazakhstan probably represent a recent spread of a limited number of plants from the primary distribution area and might be well adapted to winter low temperature.

Introduction

Wild barley, Hordeum vulgare ssp. spontaneum, is a direct progenitor of cultivated barley and valuable genetic resource for its improvement for resistance to biotic and abiotic stresses and productivity. There are multiple lines of evidence suggesting that most genetic variation of wild barley is concentrated in populations that grow in the Middle East (Nevo 1992). There are several reports describing the extent and structure of the genetic variation of wild barley populations in some parts of Central Asia (Turuspekov , Volis ), which is the marginal distribution area of this subspecies (Bothmer ). At the same time, despite the facts that Kazakhstan is the largest country in Central Asia and barley is second important cereal crop after wheat in Kazakhstan, there were no reports on characterization of the genetic diversity of wild barley grown in Kazakhstan.

The genetic diversity in wild barley populations has been studied by using a number of different genetic markers, including storage proteins (Doll and Brown 1979, Nevo ), isozymes (Brown , Nevo ), and PCR based DNA markers (Baum , Nevo , Owuor , Turpeinen ). Of these technologies, the automated genome-wide profiling of plants with single nucleotide polymorphism (SNP) markers is increasingly used for evaluation of genetic resources, including barley (Close ).

The objectives of this work were to (i) describe the area of distribution for wild barley populations growing in Kazakhstan and (ii) assess the level and structure of genetic diversity in these populations as compared to accessions from the Middle East.

Materials and Methods

The plant material from southern Kazakhstan consisted of 96 accessions of wild barley that represent 14 different populations (Table 1, Fig. 1). The plants were collected by the expedition team of Kazakhstan with distances at least 10 m apart, and locations were recorded by GPS device (Table 1). In order to make a small subset of H. vulgare ssp. spontaneum collection from International Barley Core Collection, 36 accessions were selected to reflect the structure of country of origin in total 150 accessions. Of the 36 accessions, 25 were collected from the Middle East and used for diversity comparison (Table 2). All wild barley accessions from Kazakhstan and the Middle East were grown in individual pots in a glasshouse. DNA samples were extracted and purified by using commercial kits (Qiagen, CA, USA). The DNA concentration for each sample was adjusted to 50 ng/μl. Selected 384 SNPs from the Illumina oligonucleotide pool assay (OPA) with known genetic positions from a consensus barley genetic map (Close ) were used in this study. The map location of each SNP was given according to Sato . PCR, hybridization, and scanning were performed according to the GoldenGate genotyping assay protocol (Illumina Inc.; Fan ) at the Institute of Plant Science and Resources, Okayama University, Japan. SNP basecalling was performed using GenomeStudio software (Illumina Inc.).

Table 1

Collection sites of 14 wild barley populations in southern Kazakhstan

Population	ID in Fig. 2	Longitude	Latitude	Altitude (m)	Location
1	1.1–1.7	E42 23 56	N069 38 27	620	Suburb of Shymkent
2	2.1–2.7	E42 09 28	N069 45 30	762	Near Akbastau
3	3.1–3.7	E41 57 95	N069 29 55	715	Near Kazygurt hils
4	4.1–4.7	E41 46 15	N069 32 12	699	Near Turbat
5	5.1–5.7	E41 52 34	N069 28 15	520	Near Sharapkhana
6	6.1–6.7	E41 29 16	N069 26 09	621	Near Zhibek Zholy
7	7.1–7.7	E41 29 15	N069 26 08	623	Near Zhibek Zholy
8	8.1–8.7	E41 29 15	N069 26 09	626	Near Zhibek Zholy
9	9.1–9.7	E41 29 17	N069 26 09	623	Near Zhibek Zholy
10	10.1–10.7	E41 26 20	N069 07 18	416	Near Saryagash
11	11.1–11.7	E41 24 37	N069 03 54	400	Near Abai
12	12.1–12.7	E41 12 48	N068 35 59	270	Near Birlik
13	13.1–13.7	E41 53 02	N069 35 48	780	Near Altyntobe
14	14.1–14.5	E41 29 18	N069 26 08	624	Near Krasnovodopad breeding station

Fig. 1

The location of collection sites (2008–2009) of wild barley populations in south Kazakhstan province.

Table 2

Wild barley accessions from the Middle East (small subset accessions of International Barley Core Collection)

No	ID in Fig. 2	Accession No.	Collection site	Longitude	Latitude	Altitude (m)
1	SYR1	180001	Idlib	E036 42	N36 13	320
2	JOR1	180007	Irbid	E035 55	N32 27	500
3	IRQ1	180049	Sulaymaniyah	E044 50	N35 32	640
4	PAL1	180303	(Tel Gezer)	E034 55	N31 51	n/a
5	PAL2	180982	(Atlit)	E034 56	N32 41	n/a
6	JOR2	181215	Assarieh; Wadi Al Gazira	E035 54	N32 29	690
7	SYR2	181238	Homs	E039 02	N34 45	530
8	SYR3	181306	Near Birin	E036 39	N34 59	430
9	JOR3	181393	Irbid	E035 39	N32 29	300
10	JOR4	181412	Amman	E035 39	N31 48	−220
11	JOR5	181440	Muta; S of Karak	E035 42	N31 04	1220
12	JOR6	181454	Tafila	E035 34	N30 42	1450
13	JOR7	181466	Irbid	E035 50	N32 39	530
14	SYR4	181488	Damascus	E036 32	N33 50	1500
15	LBN1	181573	Rachaiya; 1 km before Ain Hircha	E035 49	N33 27	1020
16	LBN2	181585	2 km N Kosaya road to Deir El Ghazal	E036 01	N33 48	1180
17	IRN1	181590	30 km N Urumiyeh to Salmas	E045 00	N38 05	1210
18	IRN2	181594	Arak town	E049 48	N34 05	1600
19	IRQ2	181598	20 km E Mosul to Aqrah	E043 25	N36 23	470
20	SYR5	181616	Aleppo road from Kamishli	E041 12	N37 01	480
21	LBN3	181632	2 km before Ain Arab	E035 51	N33 35	1310
22	IRN3	181652	Farm Hoseyn Abad, 116 km	E047 46	N36 16	n/a
23	TUR1	181678	Bostancik village	E037 21	N36 52	610
24	TUR2	181687	20 km to Nizip	E037 36	N37 02	745
25	SYR6	181725	Kafr Nabil; 2 km S	E036 33	N35 36	560

The statistical analyses of population genetics parameters were performed using GenAlEx (Peakall and Smouse 2006, 2012) and Popgene (Yeh and Boyle 1997). The phylogenetic trees were constructed using neighbor joining method by 1000 bootstrap replications in MEGA6 (Tamura ).

Results and Discussion

Geographic locations of wild barley population in southern Kazakhstan

Collecting trips in 2008–2009 showed that wild barley (H. vulgare ssp. spontaneum) distribution in Kazakhstan is restricted to the most southern province. The region is considered as one of the most northern margin of distribution for this wild barley by Bothmer but not described in an earlier study of Harlan and Zohary (1966). Within the province the typical area of growth is in valleys located close to mountain ranges with some sporadic appearance on roadsides of deserted areas, such as population 12 (Table 1, Fig. 1). The most northern successful collecting location was the regional capital Shymkent (population 1). The 14 populations collected were situated within the province as follows: north-east side (populations 1 and 2); south side (populations 3–11, 13 and 14); and far west-south side (population 12). Searches for additional populations to the north, east, and west from Shymkent were not successful. Therefore, assuming that wild barley migrated from the Middle East via other countries of Central Asia, e.g. Uzbekistan, Shymkent might be the most northern location of wild barley in Kazakhstan. The elevation of collection sites varied from 270 m (deserted area, population 12) to 780 m (population 13) above sea level (Table 1). Plants have winter growth habit and seed matures at the end of May under local growing conditions. The major limitation for expansion of wild populations to the north appears to be winter low temperature. Therefore, the accessions which were studied might have undergone natural selection for winter survival.

The extent and structure of wild barley populations from Kazakhstan

The genetic diversity of wild barley populations was studied based on the analysis of 384 SNP genotypes using the GoldenGate assay (Illumina Inc.). Of these SNPs, 278 with excellent base call rates for all accessions were selected for further analysis. Forty-five SNPs monomorphic across all samples were removed from the analysis. Therefore, a total of 233 SNP markers were used in the genetic analysis.

The parameters from the genetic diversity analysis of 96 accessions from 14 populations indicated a relatively low level of genetic diversity (He) with the range from 0.002 in population 6 to 0.160 in population 12 (Table 3). The highest within-population heterogeneity was observed for the most southern population (12), which was collected on the roadside of deserted area close proximity to the border with Uzbekistan (Fig. 1). Population 6, which was collected near the Krasnovodopad breeding station in southern Kazakhstan had the lowest genetic diversity. Phylogenetic analysis of accessions from all the 14 populations revealed only one cluster with five sub-clusters (Ia–Ie) as shown in Fig. 2. Although population 6 showed distribution within a sub-cluster, most of the accessions form a population were in multiple sub-clusters and the populations might share genetically close accessions. Therefore, there was a lack of correlation between geographic and genetic distances.

Table 3

Mean values of diversity parameters for 14 wild barley populations from Kazakhstan based on 233 SNP markers

Population	1	2	3	4	5	6	7	8	9	10	11	12	13	14
Number of accessions	7	7	7	7	7	7	7	7	7	7	7	7	7	5
Naa	1.313	1.378	1.292	1.206	1.339	1.009	1.373	1.232	1.300	1.391	1.408	1.446	1.343	1.232
Neb	1.148	1.208	1.142	1.067	1.241	1.003	1.272	1.161	1.175	1.264	1.255	1.275	1.207	1.169
Ic	0.151	0.194	0.142	0.084	0.197	0.004	0.221	0.139	0.159	0.226	0.222	0.241	0.185	0.139
Hed	0.096	0.127	0.091	0.050	0.134	0.002	0.152	0.095	0.105	0.153	0.148	0.160	0.123	0.095

Mean allele number.

Effective number of alleles.

Shannon-Weave information index.

Expected heterozygosity.

Fig. 2

The consensus phylogenic tree for accessions from Kazakhstan (96) and the Middle East (25) constructed using the circle style of the neighbor joining method. Numerals with period indicate population and plant numbers of accessions from Kazakhstan. Accessions of the Middle East are shown in country code and numerals as designated in Table 2. Bootstrap values with 1000 replications are shown in italics.

Comparative assessment of genetic variation in wild barley populations from the Middle East and Kazakhstan

In order to compare the diversity level of wild barley populations from Kazakhstan with that of germplasm from the center of diversity, 25 accessions from the Middle East (accessions of International Barley Core Collection) were analyzed using the same set of 233 SNP markers which were well spread on barley genome. The results of this comparative genetic description of the two germplasm arrays is summarized in Table 4. All parameters, including effective number of alleles and heterozygosity index, indicate higher levels of genetic variation for the Middle East germplasm than for the Kazakhstan germplasm. In particular, the heterozygosity parameter (He) is much higher for the Middle East germplasm (0.29) than for the Kazakhstan germplasm (0.19). The analysis of variance including all the accessions from the Middle East and Kazakhstan arrays revealed that 23% of the total variation can be attributed to the variation between arrays, while 77% of the total variation is within arrays. On the other hand, the neighbor joining phylogenetic tree clearly differentiates the accessions from the two geographic areas, although accession 5.4 from Kazakhstan population was closer to the Middle East cluster II (Fig. 2).

Table 4

Mean values for genetic diversity parameters in populations from the Middle East and Kazakhstan

Population	Middle East	Kazakhstan
Number of accessions	25	96
Naa	2.19 ± 0.03	1.65 ± 0.04
Neb	1.48 ± 0.02	1.32 ± 0.03
Ic	0.47 ± 0.01	0.28 ± 0.02
Hed	0.29 ± 0.01	0.19 ± 0.01

Mean allele number.

Effective number of alleles.

Shannon-Weave information index.

Expected heterozygosity.

An advantage of this study is that selected SNP markers were previously well-characterized and information on EST markers is available in barley databases (Close ). However, the selected 384 SNP markers were developed from EST sequences of several standard cultivars, including Haruna Nijo, Barke, and Morex (Sato ). The only wild barley accession that used as a source for EST sequences was line H602 (H. vulgare ssp. spontaneum var. transcaspicum). Therefore, polymorphisms detected in the wild barley population in this study may be biased. Nevertheless, the SNPs represent the best currently available tool for characterizing genetic diversity in barley. The high winter hardiness of wild barleys in this area (Kazakhstan and Uzbekistan) were observed at the winter barley screening nursery at Oregon State University, USA (Hayes, personal communication). Wild barley accessions in Kazakhstan probably represents a recent spread of a limited number of plants by human activity from the primary distribution area that were reported previously (Harlan and Zohary 1966). Therefore, wild barley in Kazakhstan may be one of examples for expansion of its secondary distribution area.

The fingerprinted wild barley germplasm can be a source of alleles for improvement of cultivated barley. The procedure is clearly shown by Hori who backcrossed a wild barley segment into an elite cultivar background. SNP marker information is the key for accomplishing efficient introgression. The barley genome sequence data (The International Barley Genome Sequencing Consortium 2012) will provide a larger catalog of SNPs for introducing genes from wild barley more precisely and can help to eliminate deleterious segments efficiently.